Modified alginates for anti-fibrotic materials and applications

ABSTRACT

Covalently modified alginate polymers, possessing enhanced biocompatibility and tailored physiochemical properties, as well as methods of making and use thereof, are disclosed herein. The covalently modified alginates are useful as a matrix for coating of any material where reduced fibrosis is desired, such as encapsulated cells for transplantation and medical devices implanted or used in the body.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 14/817,084 filed Aug.3, 2015, which claims priority to and benefit of U.S. ProvisionalApplication No. 62/032,148, filed Aug. 1, 2014, and U.S. ProvisionalApplication No. 62/180,415, filed on Jun. 16, 2015, which areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grants EB000244,EB000351, DE013023 and CA151884 awarded by the National Institutes ofHealth (NIH) and Grant W81XWH-13-1-0215 awarded by the Department ofDefense (DOD). The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the use of alginates, chemicallymodified to enhance their biocompatibility and anti-fibrotic properties;to their use to coat or encapsulate materials, products, and devices,such as cells, implants, and medical devices; and to methods of treatingdiseases or disorders, including diabetes, by implantation of themodified alginates and materials coated or encapsulated with themodified alginates.

BACKGROUND OF THE INVENTION

The foreign body response is an immune-mediated reaction that impactsthe fidelity of implanted biomedical devices (Anderson et al., Semin.Immunol. 20:86-100 (2008); Langer, Adv. Mater. 21:3235-3236 (2009);Ward, J. Diabetes Sci. Technol. Online 2:768-777 (2008); Harding &Reynolds, Trends Biotechnol. 32:140-146 (2014)). Macrophage recognitionof biomaterial surfaces in these devices initiate a cascade ofinflammatory events that result in the fibrous and collagenousencapsulation of these foreign materials (Anderson et al. (2008); Ward(2008); Harding & Reynolds (2014); Grainger, Nat. Biotechnol. 31:507-509(2013); Williams, Biomaterials 29:2941-2953 (2008)). This encapsulation,over time, often leads to device failure and can result in discomfortfor the recipient (Anderson et al. (2008); Harding & Reynolds (2014);Williams (2008)). These adverse outcomes emphasize the critical need forbiomaterials that do not elicit foreign body responses to overcome thiskey challenge to long-term biomedical device function.

The foreign body response to implanted biomaterials is the culminationof inflammatory events and wound-healing processes resulting in implantencapsulation (Anderson et al. (2008)). The final pathological productof this response is fibrosis, which is characterized by the accumulationof excessive extracellular matrix at sites of inflammation and is a keyobstacle for implantable medical devices as the cellular and collagenousdeposition isolate the device from the host (Anderson et al. (2008);Wick et al., Annu. Rev. Immunol. 31:107-135 (2013); Wynn & Ramalingam,Nat. Med. 18:1028-1040 (2012)). This device isolation can interfere withsensing of the host environment, lead to painful tissue distortion, cutoff nourishment (for implants containing living, cellular components),and ultimately lead to device failure. Materials commonly used formedical device manufacture today elicit a foreign body response thatresults in fibrous encapsulation of the implanted material (Langer(2009); Ward (2008); Harding & Reynolds (2014); Williams (2008); Zhanget al., Nat. Biotechnol. 31:553-556 (2013)). Overcoming the foreign bodyresponse to implanted devices could pave the way for implementing newmedical advances, making the development of materials with bothanti-inflammatory and anti-fibrotic properties a critical medical need(Anderson et al. (2008); Langer (2009); Harding & Reynolds (2014)).

Macrophages are a key component of material recognition and activelyadhere to the surface of foreign objects (Anderson et al. (2008); Ward(2008); Grainger, Nat. Biotechnol. 31:507-509 (2013); Sussman et al.,Ann. Biomed. Eng. 1-9 (2013) (doi:10.1007/s10439-013-0933-0)). Objectstoo large for macrophage phagocytosis initiate processes that result inthe fusion of macrophages into foreign-body giant cells. Thesemulti-nucleated bodies amplify the immune response by secretingcytokines and chemokines that result in the recruitment of fibroblaststhat actively deposit matrix to isolate the foreign material (Andersonet al. (2008); Ward (2008); Rodriguez et al., J. Biomed. Mater. Res. A89:152-159 (2009); Hetrick et al., Biomaterials 28:4571-4580 (2007)).This response has been described for materials of both natural andsynthetic origins that encompass a wide range of physicochemicalproperties, including alginate, chitosan, dextran, collagen, hyaluronan,poly(ethylene glycol) (PEG), poly(methyl methacrylate) (PMMA),poly(2-hydroxyethyl methacrylate) (PHEMA), polyurethane, polyethylene,silicone rubber, Teflon, gold, titanium, silica, and alumina (Ward(2008); Ratner, J. Controlled Release 78:211-218 (2002)).

The transplantation of hormone- or protein-secreting cells fromgenetically non-identical members of the same species (i.e.allotransplantation) or from other species (i.e. xenotransplantion) is apromising strategy for the treatment of many diseases and disorders.Using alginate microcapsules to provide immunoisolation, hormone- orprotein-secreting cells can be transplanted into a patient without theneed for extensive treatment with immunosuppressant drugs. Thisprinciple has been successfully demonstrated by the transplantation ofalginate-encapsulated pancreatic β-cells in diabetic rat models (Lim, F.and Sun, A. M. Science. 210, 908-910 (1980)). Methods of encapsulatingbiological material in alginate gels are described, for example, in U.S.Pat. No. 4,352,883 to Lim. In the Lim process, an aqueous solutioncontaining the biological materials to be encapsulated is suspended in asolution of a water soluble polymer. The suspension is formed intodroplets which are configured into discrete microcapsules by contactwith multivalent cations such as Ca²⁺. The surface of the microcapsulesis subsequently crosslinked with polyamino acids, forming asemipermeable membrane around the encapsulated materials.

The Lim method employs conditions which are mild enough to encapsulatecells without adversely affecting their subsequent survival andfunction. The resulting alginate microcapsules are semipermeable,possessing sufficient porosity to permit nutrients, waste, and thehormones and/or proteins secreted from encapsulated cells to diffusefreely into and out of the microcapsules, and, when implanted into ananimal host, the alginate microcapsules effectively isolate theencapsulated cells from the host's immune system. See also U.S. Pat. No.7,807,150 to Vacanti, et al.

Many other synthetic materials have been tried, including blockcopolymers such as polyethyleneglycol-diacrylate polymers,polyacrylates, and thermoplastic polymers, as reported by U.S. Pat. No.6,129,761 to Hubbell and by Aebischer, et al, J Biomech Eng. 1991 May113(2):178-83. See Lesney Modern Drug Discovery 4(3), 45-46, 49, 50(2001) for review of these materials.

Since Lim first reported on the transplantation of encapsulated cells,many other have tried to create “bioreactors” for cells that couldmaintain viability of the cells in the absence of vascularization, bydiffusion of nutrients, gases and wastes through the encapsulatingmaterials, and still protect the cells from the body's immune defensesagainst foreign cells and materials. Unfortunately, efforts to translatethese therapies into human subjects have proven difficult. For example,alginate-encapsulated porcine islet cells transplanted into a humansubject suffering from Type 1 diabetes initially demonstratedsignificant improvement and required decreased insulin dosing. However,by week 49, the patient's insulin dose retuned to pre-transplant levels(Elliot, R. B. et al. Xenotransplantation. 2007; 14(2): 157-161).

In some cases, it is desirable to elicit fibrosis, for example, when thecells are implanted as a bulking material, as described in U.S. Pat. No.6,060,053 and as subsequently approved by the Food and DrugAdministration for treatment of vesicoureteral reflux.

The diminished efficacy of the implanted cells over time is the resultof fibroblastic overgrowth of the alginate capsules. The alginate gelmatrix provokes an inflammatory response upon implantation, resulting inthe encapsulation of the alginate matrix with fibrous tissue. Thefibrous tissue on the alginate capsule surface reduces the diffusion ofnutrients and oxygen to the encapsulated cells, causing them to die. Nobetter results have been obtained with the other materials.

Therefore, it is an object of the invention to provide polymers suitablefor coating products, devices, and surfaces where the polymers havegreater long term biocompatibility following implantation of theproducts, devices, and surfaces.

It is also an object of the invention to provide polymers suitable forcoating products, devices, and surfaces where the polymers have lessforeign body response following implantation of the products, devices,and surfaces.

It is also an object of the invention to provide polymers suitable forencapsulation and implantation of cells where the polymers have greaterlong term biocompatibility following implantation.

It is also an object of the invention to provide polymers suitable forencapsulation and implantation of cells where the polymers have lessforeign body response following implantation.

It is also an object of the invention to provide chemically modified,ionically crosslinkable alginates with improved biocompatibility andtailored physiochemical properties, including gel stability, pore size,and hydrophobicity/hydrophilicity.

It is also an object of the invention to provide chemically modified,ionically crosslinkable alginates with less foreign body response.

It is also an object of the invention to provide methods for the coatingof products, devices, and surfaces using modified alginate polymers.

It is also an object of the invention to provide methods for theencapsulation of cells using modified alginate polymers.

It is also an object of the invention to provide methods for treating adisorder or disease in a human or animal patient by transplanting orimplanting products, devices, and surfaces coated with a modifiedalginate polymer.

It is also an object of the invention to provide methods for treating adisorder or disease in a human or animal patient by transplantingexogenous biological material encapsulated in a modified alginatepolymer.

It is also an object of the invention to provide high-throughput methodsfor the characterization of modified alginate polymers.

SUMMARY OF THE INVENTION

Alginates, chemically modified to tailor their biocompatibility andphysical properties, have been developed. The modified alginatesdescribed herein provide enhanced properties relative to unmodifiedalginates. Moreover, based on the discovery that the starting materials,as well as chemically modified and reacted materials, must beexhaustively purified to remove contaminants prior to implantation toprevent encapsulation, these materials are less likely to elicit fibrouscapsule formation following implantation.

In some embodiments, modified alginates are alginate polymers thatcontain one or more covalently modified monomers defined by Formula I

wherein,

X is oxygen, sulfur, or NR₄;

R₁ is hydrogen, or an organic grouping containing any number of carbonatoms, preferably 1-30 carbon atoms, more preferably 1-20 carbon atoms,more preferably 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative R₁ groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, sulfonyl,substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl,phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl,C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup;

Y₁ and Y₂ independently are hydrogen or —PO(OR₅)₂; or

Y₂ is absent, and Y₁, together with the two oxygen atoms to which Y₁ andY₂ are attached form a cyclic structure as shown in Formula II

wherein; and

R₂ and R₃ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, preferably 1-30 carbon atoms, morepreferably 1-20 carbon atoms, more preferably 1-14 carbon atoms, andoptionally including one or more heteroatoms such as oxygen, sulfur, ornitrogen grouping in linear, branched, or cyclic structural formats,representative R₂ and R₃ groupings being alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy,aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup; or

R₂ and R₃, together with the carbon atom to which they are attached,form a 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and

R₄ and R₅ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, preferably 1-30 carbon atoms, morepreferably 1-20 carbon atoms, more preferably 1-14 carbon atoms, andoptionally including one or more heteroatoms such as oxygen, sulfur, ornitrogen grouping in linear, branched, or cyclic structural formats,representative R₄ and R₅ groupings being alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy,aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup.

In some embodiments, modified alginates are alginate polymers thatcontain one or more covalently modified monomers defined by Formula I

wherein,

X is oxygen, sulfur, or NR₄;

R₁ is, independently in the one or more modified monomers

or —R₆—R^(b), wherein a is an integer from 1 to 30, z is an integer from0 to 5, n is an integer from 1 to 12, m is an integer from 3 to 16, andR^(a) and R^(b) are independently selected from alkoxy, amino,alkylamino, dialkylamino, hydroxy, alkenyl, alkynyl, substituted alkyl,substituted alkenyl, substituted alkynyl, phenyl, substituted phenyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, substitutedalkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy,alkylthio, substituted alkylthio, phenylthio, substituted phenylthio,arylthio, substituted arylthio, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; ortogether with the carbon atom to which they are attached, form a 3- to8-membered unsubstituted or substituted carbocyclic or heterocyclicring;wherein

Y₁ and Y₂ independently are hydrogen or —PO(OR₅)₂; or

Y₂ is absent, and Y₁, together with the two oxygen atoms to which Y₁ andY₂ are attached form a cyclic structure as shown in Formula II

wherein

R₂ and R₃ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, 1-30 carbon atoms, 1-20 carbon atoms, or1-14 carbon atoms, and optionally including one or more heteroatoms suchas oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclicstructural formats, representative R₂ and R₃ groupings being alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group; or

R₂ and R₃, together with the carbon atom to which they are attached,form a 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and

R₄, R₅, R₆, R₈, and R₉ are, independently, hydrogen or an organicgrouping containing any number of carbon atoms, 1-30 carbon atoms, 1-20carbon atoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, modified alginates are alginate polymers thatcontain one or more covalently modified monomers defined by Formula I

wherein,

X is oxygen, sulfur, or NR₄;

R₁ is, independently in the one or more modified monomers,

wherein k is an integer from 1 to 10; wherein z is an integer from 0 to5; wherein w is an integer from 0 to 4; wherein X_(d) is absent, O or S;

wherein R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₃₉, R₄₀, and R₄₁ areindependently hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group;

wherein R₃₇ is C or Si;

wherein X_(g) and R₃₈ are independently alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkylene, substituted alkylene, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, sulfonyl,substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl,phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl,C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup; and

wherein R^(a) and R^(c) are independently alkoxy, amino, alkylamino,dialkylamino, hydroxy, alkenyl, alkynyl, substituted alkyl, substitutedalkenyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, substitutedalkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy,alkylthio, substituted alkylthio, phenylthio, substituted phenylthio,arylthio, substituted arylthio, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; ortogether with the carbon atom to which they are attached, form a 3- to8-membered unsubstituted or substituted carbocyclic, heterocyclic ringor

wherein R₈, R₉, or both are, independently, hydrogen, alkyl, substitutedalkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxy, alkenyl,alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl,carbonyl, substituted carbonyl, carbinol,

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group;

wherein y is an integer from 0 to 11; wherein R^(d) and R^(e) are eachindependently alkoxy, amino, alkylamino, dialkylamino, hydroxy, alkenyl,alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl,phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, andpolypeptide group; or together with the carbon atom to which they areattached, form a 3- to 8-membered unsubstituted or substitutedcarbocyclic or heterocyclic ring;

wherein R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, and R₂₃ are independently C, O, N, orS, wherein the bonds between adjacent R₁₈ to R₂₃ are double or singleaccording to valency, and wherein R₁₈ to R₂₃ are bound to none, one, ortwo hydrogens according to valency; and

wherein R₂₄ is independently —(CR₂₅R₂₅)_(p)— or—(CR₂₅R₂₅)_(p)—X_(b)—(CR₂₅R₂₅)_(q)—, wherein p and q are independentlyintegers from 0 to 5, wherein X_(b) is absent, —O—, —S—, —SO₂—, or NR₄,wherein each R₂₅ is independently absent, hydrogen, ═O, ═S, —OH, —SH,—NR₄, wherein R₄ is alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group;

wherein R₈, and R₉ are not both hydrogen; wherein at least one R^(b) orR^(c) is defined by Formula XIII;

wherein

Y₁ and Y₂ independently are hydrogen or —PO(OR₅)₂; or

Y₂ is absent, and Y₁, together with the two oxygen atoms to which Y₁ andY₂ are attached form a cyclic structure as shown in Formula II

wherein

R₂ and R₃ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, 1-30 carbon atoms, 1-20 carbon atoms, or1-14 carbon atoms, and optionally including one or more heteroatoms suchas oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclicstructural formats, representative R₂ and R₃ groupings being alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group; or

R₂ and R₃, together with the carbon atom to which they are attached,form a 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and

R₄ and R₅ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, 1-30 carbon atoms, 1-20 carbon atoms, or1-14 carbon atoms, and optionally including one or more heteroatoms suchas oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclicstructural formats, representative organic groupings being alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group.

In some embodiments, y in Formula IX is an integer from 0-3; R^(e) isindependently amino, hydroxyl, thiol, oxo, or substituted orunsubstituted C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, or C₁-C₆alkylthio;

where R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, and R₂₃ are independently C, O, N, or S,where the bonds between adjacent R₁₈ to R₂₃ are double or singleaccording to valency, and where R₁₈ to R₂₃ are bound to none, one, ortwo hydrogens according to valency; and

where R₂₄ is independently —(CR₂₅R₂₅)_(p)— or—(CR₂₅R₂₅)_(p)—X_(b)—(CR₂₅R₂₅)_(q)—, where p and q are independentlyintegers from 0 to 3, where X_(b) is absent, —O—, —S—, —SO₂—, or NR₄,where each R₂₅ is independently absent, hydrogen, ═O, ═S, —OH, —SH,—NR₄, where R₄ is substituted or unsubstituted C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ alkylamino, or C₁-C₆ alkylthio.

In some embodiments, y in Formula IX is 2, R₁₈ is N, R₁₉, R₂₀, R₂₁, R₂₂,or R₂₃ is S, both R^(e) are oxo and are bonded to the S, and all of thebonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments, y in Formula IX is 2, both R^(e) are oxo and arebonded to R₂₁, R₁₈ is N, R₂₁ is S, and all of the bonds between adjacentR₁₈ to R₂₃ are single.

In some embodiments, y in Formula IX is 2, both R^(e) are oxo and arebonded to R₂₁, R₁₈ is N, R₂₁ is S, and all of the bonds between adjacentR₁₈ to R₂₃ are single, X_(b) is absent, q is 0, p is 1, and each R₂₅ ishydrogen.

In some embodiments, y in Formula IX is 1, R^(e) is amino, and three ofthe bonds between adjacent R₁₈ to R₂₃ are double and three of the bondsbetween adjacent R₁₈ to R₂₃ are single.

In some embodiments, y in Formula IX is 1, R^(e) is amino and is bondedto R₂₁, and three of the bonds between adjacent R₁₈ to R₂₃ are doubleand three of the bonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments, y in Formula IX is 1, R^(e) is amino and is bondedto R₂₁, and three of the bonds between adjacent R₁₈ to R₂₃ are doubleand three of the bonds between adjacent R₁₈ to R₂₃ are single, X_(b) isabsent, p is 0 and q is 0.

In some embodiments, y in Formula IX is 0, R₁₉, R₂₀, R₂₁, R₂₂, or R₂₃ isO, and all of the bonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments, y in Formula IX is 0, R₁₉ is O, and all of thebonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments, y in Formula IX is 0, R₁₉ is O, all of the bondsbetween adjacent R₁₈ to R₂₃ are single, X_(b) is oxygen, p is 1, q is 0and each R₂₅ is hydrogen.

In some embodiments, R₁₉ and R₂₃ of Formula IX are O and all of thebonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments, R₁₉ and R₂₃ of Formula IX are O, the bonds betweenR₁₈ and R₁₉, and between R₂₁ and R₂₂ are double bonds, and the rest ofthe bonds in the ring are single bonds.

In some embodiments, y in Formula IX is 1, R^(e) is alkoxy and is bondedto R₁₉, R₂₀, R₂₁, R₂₁, R₂₂, or R₂₃, three of the bonds between adjacentR₁₈ to R₂₃ are double and three of the bonds between adjacent R₁₈ to R₂₃are single.

In some embodiments, y in Formula IX is 1, R^(e) is alkoxy and is bondedto R₁₉, three of the bonds between adjacent R₁₈ to R₂₃ are double andthree of the bonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments, y in Formula IX is 1, R^(e) is methoxy, and isbonded to R₁₉, R₁₈ to R₂₃ are carbon atoms, three of the bonds betweenadjacent R₁₈ to R₂₃ are double and three of the bonds between adjacentR₁₈ to R₂₃ are single, X_(b) is absent, p is 0 and q is 0.

In some embodiments, y in Formula IX is 1, R^(e) is hydroxyl.

In some embodiments, y in Formula IX is 1 and R^(e) is hydroxyl bondedat the position para- to the methylene group.

In some embodiments, y in Formula IX is 1, R^(e) is Formula XIII shownbelow:

wherein R₈ is a substituted alkyl and R₉ is a dialkylamino, or R₈ is adialkylamino and R₉ is a substituted alkyl, wherein the substitutedalkyl is hydroxymethyl and the dialkylamino is N,N-diethylamino.

In some embodiments, y in Formula IX is 1, R^(e) is Formula XIII,wherein R₈ is hydrogen and R₉ is Formula IX shown below:

or R₈ is Formula IX and R₉ is hydrogen. In some embodiments, y inFormula IX is 0, R₁₉, R₂₀, R₂₁, R₂₂, or R₂₃ is O, and all of the bondsbetween adjacent R₁₈ to R₂₃ are single. In some embodiments, y inFormula IX is 0, R₁₉ is O, and all of the bonds between adjacent R₁₈ toR₂₃ are single. In some embodiments, y in Formula IX is 0, R₁₉ is O, allof the bonds between adjacent R₁₈ to R₂₃ are single, X_(b) is oxygen, pis 1, q is 0 and each R₂₅ is hydrogen.

In some embodiments, y in Formula IX is 1, R^(e) is Formula XIII,wherein R₈ is hydrogen and R₉ is Formula VII or Formula VIII shownbelow:

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments R₃₁ of Formula VII is alkyl. In some embodiments,R₃₁ is methyl.

In some embodiments R₃₁ of Formula VII is methyl, R₃₂ and R₃₃ arehydrogen.

In some embodiments, y in Formula IX is 1 and R^(e) is hydroxyl bondedat the position para- to the methylene group.

In some embodiments of Formula XII, k is 1 and R^(c) is hydroxyl.

In some embodiments of Formula XII, k is 1, R^(c) is hydroxyl, and X_(d)is absent.

In some embodiments of Formula XII, k is 1, R^(c) is hydroxyl, X_(d) isabsent, and R₁₀—R₁₇ are hydrogen.

In some embodiments of Formula XII, R^(c) is alkoxy.

In some embodiments of Formula XII, R^(c) is methoxy and X_(d) is O.

In some embodiments of Formula XII, R^(c) is methoxy, X_(d) is O, andR₁₀—R₁₇ are hydrogen.

In some embodiments of Formula XII, k is 2 and R^(c) is alkylamino.

In some embodiments of Formula XII, k is 2, R^(c) is methylamino, andX_(d) is absent.

In some embodiments of Formula XII, k is 2, R^(c) is methylamino, X_(d)is absent, and R₁₀—R₁₇ are hydrogen.

In some embodiments of Formula XII, k is 3, X_(d) is O and R^(c) isFormula XIII shown below:

wherein R₈ and R₉ are alkyl.

In some embodiments of Formula XII, k is 3, X_(d) is O, and R^(c) isFormula XIII, wherein R₈ and R₉ are methyl.

In some embodiments of Formula XII, k is 3, X_(d) is O and R^(c) isFormula XIII, wherein R₈ is hydrogen, and R₉ is carbonyl, or R₈ iscarbonyl, and R₉ is hydrogen.

In some embodiments of Formula XII, k is 3, X_(d) is O and R^(c) isFormula XIII, wherein R₈ is hydrogen, and R₉ is acetyl, or R₈ is acetyl,and R₉ is hydrogen.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII, R₈is hydrogen, and R₉ is Formula IX shown below:

or R₈ is hydrogen and R₉ is Formula IX.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 2, R₁₈ is N, R₁₉, R₂₀, R₂₁,R₂₂, or R₂₃ is S, both R^(e) are oxo and are bonded to the S, and all ofthe bonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 2, both R^(e) are oxo and arebonded to R₂₁, R₁₈ is N, R₂₁ is S, and all of the bonds between adjacentR₁₈ to R₂₃ are single.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 2, both R^(e) are oxo and arebonded to R₂₁, R₁₈ is N, R₂₁ is S, and all of the bonds between adjacentR₁₈ to R₂₃ are single, X_(b) is absent, q is 0, p is 1, and each R₂₅ ishydrogen.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 1, R^(e) is amino, and threeof the bonds between adjacent R₁₈ to R₂₃ are double and three of thebonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 1, R^(e) is amino and isbonded to R₂₁, and three of the bonds between adjacent R₁₈ to R₂₃ aredouble and three of the bonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 1, R^(e) is amino and isbonded to R₂₁, and three of the bonds between adjacent R₁₈ to R₂₃ aredouble and three of the bonds between adjacent R₁₈ to R₂₃ are single,X_(b) is absent, p is 0 and q is 0.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 0, R₁₉, R₂₀, R₂₁, R₂₂, or R₂₃is O, and all of the bonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 0, R₁₉ is O, and all of thebonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments of Formula XII, k is 3 and R is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 0, R₁₉ is O, all of the bondsbetween adjacent R₁₈ to R₂₃ are single, X_(b) is oxygen, p is 1, q is 0and each R₂₅ is hydrogen.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 1, R^(e) is alkoxy and isbonded to R₁₉, R₂₀, R₂₁, R₂₁, R₂₂, or R₂₃, three of the bonds betweenadjacent R₁₈ to R₂₃ are double and three of the bonds between adjacentR₁₈ to R₂₃ are single.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 1, R^(e) is alkoxy and isbonded to R₁₉, three of the bonds between adjacent R₁₈ to R₂₃ are doubleand three of the bonds between adjacent R₁₈ to R₂₃ are single.

In some embodiments of Formula XII, k is 3 and R^(c) is Formula XIII,wherein R₈ is hydrogen, and R₉ is Formula IX, or R₈ is hydrogen and R₉is Formula IX, wherein y in Formula IX is 1, R^(e) is alkoxy such asmethoxy, and is bonded to R₁₉, R₁₈ to R₂₃ are carbon atoms, three of thebonds between adjacent R₁₈ to R₂₃ are double and three of the bondsbetween adjacent R₁₈ to R₂₃ are single, X_(b) is absent, p is 0 and q is0.

In some embodiments, y in Formula XIV is 0, R₁₉, R₂₀, R₂₁, R₂₁, or R₂₂is O, and, as valency permits, two of the bonds between adjacent R₁₈ toR₂₂ are double bonds, and three of the bonds between adjacent R₁₈ to R₂₂are single bonds.

In some embodiments, y in Formula XIV is 0, R₁₉ is O, R₁₈, R₂₀, R₂₁ andR₂₂ are C, the bonds between R₁₈ and R₂₂, and between R₂₀ and R₂₁, aredouble bonds, and the rest of the bonds in the ring are single bonds.

In some embodiments, y in Formula XIV is 0, R₁₉ is O, R₁₈, R₂₀, R₂₁ andR₂₂ are C, the bonds between R₁₈ and R₂₂, and between R₂₀ and R₂₁, aredouble bonds, the rest of the bonds in the ring are single bonds, X_(b)is absent, p is 1, q is 0, and each R₂₅ is hydrogen.

In some embodiments, y in Formula XIV is 0, R₁₉, R₂₀, R₂₁, or R₂₂ is O,and, the bonds between adjacent R₁₈ to R₂₂ are single bonds.

In some embodiments, y in Formula XIV is 0, R₁₉ is O, R₁₈, R₂₀, R₂₁ andR₂₂ are C, and the bonds between adjacent R₁₈ to R₂₂ are single bonds.

In some embodiments, y in Formula XIV is 0, R₁₉ is O, R₁₈, R₂₀, R₂₁ andR₂₂ are C, the bonds between adjacent R₁₈ to R₂₂ are single bonds, X_(b)is absent, p is 1, q is 0, and each R₂₅ is hydrogen.

In some embodiments, y in Formula XIV is 0, R₁₉ and R₂₂ are O, and thebonds between adjacent R₁₁ to R₂₂ are single bonds.

In some embodiments, y in Formula XIV is 0, R₁₉ and R₂₂ are O, R₁₈, R₂₁and R₂₂ are C, the bonds between adjacent R₁₈ to R₂₂ are single bonds.

In some embodiments, y in Formula XIV is 0, R₁₉ and R₂₂ are O, R₁₈, R₂₁and R₂₂ are C, the bonds between adjacent R₁₈ to R₂₂ are single bonds,X_(b) is absent, p is 1, q is 0, and each R₂₅ is hydrogen.

In some embodiments R₃₇ of Formula XV is Si, and X_(g) is alkynyl.

In some embodiments R₃₇ of Formula XV is Si, X_(g) is ethynyl, and R₃₈is alkylene.

In some embodiments R₃₇ of Formula XV is Si, X_(g) is ethynyl, R₃₈ ismethylene, and R₃₉, R₄₀, and R₄₁ are alkyl.

In some embodiments R₃₇ of Formula XV is Si, X_(g) is ethynyl, R₃₈ ismethylene, and R₃₉, R₄₀, and R₄₁ are methyl.

Modified alginates can be either singularly modified alginate polymersor multiply modified alginate polymers. Singularly modified alginatepolymers are alginate polymers that contain one or more covalentlymodified monomers, wherein substantially all of the covalently modifiedmonomers possess the same covalent modification (i.e. the polymercontains one ‘type’ or species of covalently modified monomer). Multiplymodified alginate polymers are alginate polymers that contain covalentlymodified monomers, wherein substantially all of the covalently modifiedmonomers do not possess the same covalent modification (i.e. the polymercontains two or more ‘types’ or species of covalently modifiedmonomers).

In some embodiments, the modified alginate polymer is a singularlymodified alginate polymer. In some embodiments, the modified alginatepolymer is one of the singularly modified alginate polymers shown below:

In preferred embodiments, the modified alginate polymer is a multiplymodified alginate polymer possessing a polysaccharide backbonecontaining mannuronate monomers, guluronate monomers, a first species ortype of covalently modified monomer defined by Formula I, and a secondspecies or type of covalently modified monomer defined by Formula I. Insome embodiments, the modified alginate polymer is one of the multiplymodified alginate polymers shown below.

In some embodiments, multiply modified alginates are alginate polymersthat contain one or more covalently modified monomers having a structureaccording to Formula III

wherein

X is oxygen, sulfur, or NR₄;

R₁, R₆, R₇, R₈, and R₉ are, independently, hydrogen or an organicgrouping containing any number of carbon atoms, 1-30 carbon atoms, 1-20carbon atoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, sulfonyl,substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl,phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl,C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup;

wherein

Y₁ and Y₂ independently are hydrogen or —PO(OR₅)₂; or

Y₂ is absent, and Y₁, together with the two oxygen atoms to which Y₁ andY₂ are attached form a cyclic structure as shown in Formula IV

wherein

R₂ and R₃ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, 1-30 carbon atoms, 1-20 carbon atoms, or1-14 carbon atoms, and optionally including one or more heteroatoms suchas oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclicstructural formats, representative R₂ and R₃ groupings being alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group; or

R₂ and R₃, together with the carbon atom to which they are attached,form a 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and

R₄ and R₅ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, 1-30 carbon atoms, 1-20 carbon atoms, or1-14 carbon atoms, and optionally including one or more heteroatoms suchas oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclicstructural formats, representative R₄ and R₅ groupings being alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group.

In some embodiments, R₈, R₉, or both are, independently, hydrogen,

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, R₁ is

wherein k is independently an integer from 1 to 30; wherein z is aninteger from 0 to 4; wherein X_(d) is O or S; wherein R₁₀, R₁₁, R₁₂,R₁₃, R₁₄, R₁₅, R₁₆, and R₁₇ are independently hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, sulfonyl, substitutedsulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; andwherein R^(a) and R^(c) are independently alkoxy, amino, alkylamino,dialkylamino, hydroxy, alkenyl, alkynyl, substituted alkyl, substitutedalkenyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, substitutedalkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy,alkylthio, substituted alkylthio, phenylthio, substituted phenylthio,arylthio, substituted arylthio, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; ortogether with the carbon atom to which they are attached, form a 3- to8-membered unsubstituted or substituted carbocyclic, heterocyclic ringor

wherein R₈, R₉, or both are, independently, hydrogen,

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, the modified alginates are alginate polymers thatcontain one or more covalently modified alginates units described byFormula I, Formula II, Formula III, or Formula IV, wherein for eachformula R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are independently hydrogen oran organic grouping containing any number of carbon atoms, 1-30 carbonatoms, 1-20 carbon atoms, or 1-14 carbon atoms, and optionally includingone or more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative organicgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and wherein R₁ is not hydrogen or an organic groupingcontaining any number of carbon atoms, 1-30 carbon atoms, 1-20 carbonatoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, the modified alginates are alginate polymers thatcontain one or more covalently modified alginates units described byFormula I, Formula II, Formula III, or Formula IV, wherein for eachformula R₁, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are independently hydrogen oran organic grouping containing any number of carbon atoms, 1-30 carbonatoms, 1-20 carbon atoms, or 1-14 carbon atoms, and optionally includingone or more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative organicgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and wherein R₂ is not hydrogen or an organic groupingcontaining any number of carbon atoms, 1-30 carbon atoms, 1-20 carbonatoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, the modified alginates are alginate polymers thatcontain one or more covalently modified alginates units described byFormula I, Formula II, Formula III, or Formula IV, wherein for eachformula R₁, R₂, R₄, R₅, R₆, R₇, R₈, and R₉ are independently hydrogen oran organic grouping containing any number of carbon atoms, 1-30 carbonatoms, 1-20 carbon atoms, or 1-14 carbon atoms, and optionally includingone or more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative organicgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and wherein R₃ is not hydrogen or an organic groupingcontaining any number of carbon atoms, 1-30 carbon atoms, 1-20 carbonatoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, the modified alginates are alginate polymers thatcontain one or more covalently modified alginates units described byFormula I, Formula II, Formula III, or Formula IV, wherein for eachformula R₁, R₂, R₃, R₅, R₆, R₇, R₈, and R₉ are independently hydrogen oran organic grouping containing any number of carbon atoms, 1-30 carbonatoms, 1-20 carbon atoms, or 1-14 carbon atoms, and optionally includingone or more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative organicgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and wherein R₄ is not hydrogen or an organic groupingcontaining any number of carbon atoms, 1-30 carbon atoms, 1-20 carbonatoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, the modified alginates are alginate polymers thatcontain one or more covalently modified alginates units described byFormula I, Formula II, Formula III, or Formula IV, wherein for eachformula R₁, R₂, R₃, R₄, R₆, R₇, R₈, and R₉ are independently hydrogen oran organic grouping containing any number of carbon atoms, 1-30 carbonatoms, 1-20 carbon atoms, or 1-14 carbon atoms, and optionally includingone or more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative organicgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and wherein R₅ is not hydrogen or an organic groupingcontaining any number of carbon atoms, 1-30 carbon atoms, 1-20 carbonatoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, the modified alginates are alginate polymers thatcontain one or more covalently modified alginates units described byFormula I, Formula II, Formula III, or Formula IV, wherein for eachformula R₁, R₂, R₃, R₄, R₅, R₇, R₈, and R₉ are independently hydrogen oran organic grouping containing any number of carbon atoms, 1-30 carbonatoms, 1-20 carbon atoms, or 1-14 carbon atoms, and optionally includingone or more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative organicgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and wherein R₆ is not hydrogen or an organic groupingcontaining any number of carbon atoms, 1-30 carbon atoms, 1-20 carbonatoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, the modified alginates are alginate polymers thatcontain one or more covalently modified alginates units described byFormula I, Formula II, Formula III, or Formula IV, wherein for eachformula R₁, R₂, R₃, R₄, R₅, R₆, R₈, and R₉ are independently hydrogen oran organic grouping containing any number of carbon atoms, 1-30 carbonatoms, 1-20 carbon atoms, or 1-14 carbon atoms, and optionally includingone or more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative organicgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and wherein R₇ is not hydrogen or an organic groupingcontaining any number of carbon atoms, 1-30 carbon atoms, 1-20 carbonatoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, the modified alginates are alginate polymers thatcontain one or more covalently modified alginates units described byFormula I, Formula II, Formula III, or Formula IV, wherein for eachformula R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₉ are independently hydrogen oran organic grouping containing any number of carbon atoms, 1-30 carbonatoms, 1-20 carbon atoms, or 1-14 carbon atoms, and optionally includingone or more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative organicgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and wherein R₈ is not hydrogen or an organic groupingcontaining any number of carbon atoms, 1-30 carbon atoms, 1-20 carbonatoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, the modified alginates are alginate polymers thatcontain one or more covalently modified alginates units described byFormula I, Formula II, Formula III, or Formula IV, wherein for eachformula R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently hydrogen oran organic grouping containing any number of carbon atoms, 1-30 carbonatoms, 1-20 carbon atoms, or 1-14 carbon atoms, and optionally includingone or more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative organicgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and wherein R₉ is not hydrogen or an organic groupingcontaining any number of carbon atoms, 1-30 carbon atoms, 1-20 carbonatoms, or 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative organic groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

Modified alginate polymers can contain any ratio of mannuronatemonomers, guluronate monomers, and covalently modified monomers. Inpreferred embodiments, greater than 5%, greater than 10%, greater than15%, greater than 20%, more preferably greater than 25%, and mostpreferably greater than 30%, of the monomers in the modified alginatepolymer are covalently modified monomers.

In preferred embodiments, the modified alginate polymer can be ionicallycrosslinked to form hydrogels using a polyvalent ion, such as Ca²⁺,Sr²⁺, or Ba². The ability of modified alginates to form stable hydrogelsin physiological conditions can be quantified using the hydrogelformation assay described herein. In preferred embodiments, the modifiedalginate polymer forms hydrogels such that the fluorescence intensitymeasured using the high throughput assay described herein is between15,000 and 55,000, preferably between 20,000 and 55,000, more preferablybetween 25,000 and 55,000.

In preferred embodiments, the modified alginate is biocompatible, andinduces a lower foreign body response than unmodified alginate. Thebiocompatibility of modified alginates can be quantitatively determinedusing in vitro and in vivo assays known in the field, including the invivo biocompatibility assay described herein. In preferred embodiments,the modified alginate polymer is biocompatible such that thefluorescence response normalized to unmodified alginate measured usingthe in vivo biocompatibility assay described herein is less than 75%,70%, 65%, 60%, 55%, or 50%. Also described are assays for thecharacterization of modified alginate polymers.

A high throughput assay useful to characterize the ability of modifiedalginate polymers to form hydrogels is also described. In someembodiments, the hydrogel formation assay described herein is used toquantify the stability of hydrogels formed from alginates or modifiedalginates. In preferred embodiments, the hydrogel formation assaydescribed herein is used as a screening tool to identify modifiedalginates capable of forming stable hydrogels. The high throughput invivo biocompatibility assay described herein is used to identifymodified alginates which induce a lower foreign body response thanunmodified alginate. Assays are also provided for quantifying thebiocompatibility of modified alginates.

Further described herein are methods of coating medical products,devices, and surfaces using modified alginate polymers. In particularembodiments, the modified alginate polymers described herein are used tocoat products, devices, and surfaces for use in methods of treating adisease or disorder in a human or animal patient. In some embodiments, adisease or disorder in a human or animal patient is treated bytransplanting or implanting products, devices, and surfaces coated witha modified alginate polymer. In particular embodiments, a disease ordisorder in a human or animal patient is treated by transplanting orimplanting products, devices, and surfaces coated with a modifiedalginate polymer.

Further described herein are methods of encapsulating biologicalmaterials using modified alginate polymers. In particular embodiments,the modified alginate polymers described herein are used to encapsulatecells for use in methods of treating a disease or disorder in a human oranimal patient. In some embodiments, a disease or disorder in a human oranimal patient is treated by transplanting exogenous biological materialencapsulated in a modified alginate polymer. In particular embodiments,a disease or disorder in a human or animal patient is treated bytransplanting cells encapsulated in a modified alginate polymer. In amore particular embodiment, diabetes is treated by transplantingpancreatic islet cells encapsulated in a modified alginate polymer.

Cells suitable for encapsulation and transplantation are preferablysecretory or metabolic cells (i.e., they secrete a therapeutic factor ormetabolize toxins, or both) or structural cells (e.g., skin, muscle,blood vessel), or metabolic cells (e.g., they metabolize toxicsubstances). In some embodiments, the cells are naturally secretory,such as islet cells that naturally secrete insulin, or naturallymetabolic, such as hepatocytes that naturally detoxify and secrete. Insome embodiments, the cells are bioengineered to express a recombinantprotein, such as a secreted protein or metabolic enzyme. Depending onthe cell type, the cells may be organized as single cells, cellaggregates, spheroids, or even natural or bioengineered tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general structure of the modified alginates obtainedusing the combinatorial synthetic approach described in Example 1. Thenumber of alginates prepared with each general structure is indicatedbelow.

FIG. 2 is a plot obtained from the hydrogel formation assay described inExample 2. The average fluorescence intensity values measured formodified alginates are plotted. Modified alginates yielding fluorescencevalues below 15,000 were considered unusable for applications wherehydrogel formation is critical (i.e. the encapsulation of cells).

FIG. 3 is a plot showing the effect of selected modified alginates onHeLa cell line viability as compared to the positive control (noalginate). Alginate (Alg) has a viability of 53%. Several polymers areshown to be more cytotoxic than Alg, however, the majority of thelibrary performs as well or better than Alg.

FIG. 4 is a plot obtained using the in vivo method described in Example5, which quantifies the biocompatibility of selected modified alginates.The fluorescence response obtained for the modified alginates using thein vivo method described in Example 5 was normalized to the fluorescenceresponse measured using unmodified alginate in order to quantify thebiocompatibility of the modified alginates in terms of % fluorescenceresponse.

FIG. 5 is a plot detailing the blood glucose level of mice transplantedwith rat islets encapsulated in selected modified alginates as well astwo different unmodified alginates (CMIT and CJOS). The dashed linerepresents normoglycemia in mice. At 5 days post-implantation, 287_F4,CJOS, 287_B4, 263_C12, and CMIT are above the dashed line while theothers are below the line. At 20 5 days post-implantation, the linesare, from top to bottom: CJOS, 287_G3, 287_F4, 287B_B4, CMIT, 287B_B8,263_C12, 263_C6, 287_B3, 287_D3, and 263_A7.

FIG. 6 is a bar graph showing inflammatory response (as measured byfluorescence normalized to VLVG) as a function of modified alginate(combined with unmodified alginate).

FIG. 7 is a diagram of the structures of amines, alcohols, azides, andalkynes used for the chemical modification of alginate. “N” designationindicates amidation reagents, “O” designations indicate esterificationreagents, and “Y” designations indicate click reagents.

FIG. 8 is a graph of FACS analysis of macrophages (CD11b+, CD68+) andneutrophils (CD11b+, Ly6 g+) isolated from Z2-Y12, Z1-Y15, Z1-Y19,SLG20, and V/S capsules retrieved after 14 days in the IP space ofC57BL/6 mice. ***=p<0 0001, ns=not significant.

FIG. 9 is a diagram of the scheme for the synthesis of 774 alginateanalogues.

FIG. 10 is a graph of Western blot quantification of α-SMA proteinisolated from implants retrieved from the STZ-C57BL/6J.

FIG. 11 is a graph showing secondary cathepsin evaluation of 70 topmodified alginates from the initial screen formulated as 300 μmcapsules. Data normalized to the fluorescence of VLVG capsules. The tenalginate analogue capsules with the lowest cathepsin levels are on theright with lighter shading.

FIG. 12 is a graph of cytokine panel analysis (Elispot) of proteinextracted from 300 μm capsules of the top ten alginate analogue capsulesand control alginate capsules (SLG20, V/S) retrieved from the IP spaceof C57BL/6 mice after 14 days. For each cohort n=5. # indicate asignificance difference between the means with p<0.01.

FIG. 13 is a chemical structure of triazole-thiomorpholine dioxide(TMTD) alginate.

FIG. 14 is graphs of FACS analysis of encapsulated human cell implantsretrieved after 14 days IP in C57BL/6 showing macrophages andneutrophils.

FIG. 15 is graphs of FACS analysis of encapsulated human cell implantsretrieved after 14 days IP in C57BL/6 showing B cells and CD8 T cells.

FIG. 16 is a heat map of proteomic quantification of proteins detectedin protein isolates from implants retrieved from the STZ-C57BL/6J. Eachcolumn in the heatmap is an individual STZ-C57BL/6 mouse from therespective cohort.

DETAILED DESCRIPTION OF THE INVENTION

Alginates are a class of linear polysaccharide copolymers formed from1-4-glycosidically linked β-D-mannuronate (M) and its C-5 epimerα-L-guluronate (G). Alginates are naturally occurring biopolymersproduced by a variety of organisms, including marine brown algae and atleast two genera of bacteria (Pseudomonas and Azotobacter). Typically,commercial alginates are isolated from marine algae, includingMacrocystis pyrifera, Ascophyllum nodosum, and various types ofLaminaria.

Three types of primary structure define the polysaccharide backbone ofalginates: homopolymeric regions of consecutive guluronate monomers(G-blocks), homopolymeric regions of consecutive mannuronate monomers(M-blocks), and regions containing alternating mannuronate andguluronate monomers (MG-blocks). The monomer blocks possess differentconformations in solution, ranging from a flexible extended structure(M-blocks) to a rigid compact structure (G-blocks). In the case ofG-blocks, the compact conformation facilitates the chelation ofmultivalent ions, notably Ca²⁺ ions, such that G-blocks in one alginatechain can be ionically crosslinked with G-blocks in another alginatechain, forming stable gels. As a result, the proportion, length, anddistribution of the monomer blocks influence the physiochemicalproperties of the alginate polymer.

In the case of commercially produced alginates obtained from algae, themolecular weight, primary structure, and overall molar ratio of uronicacid monomers (M/G ratio) in the alginate polymer depends on a number offactors, including the species producing the alginate, the time of yearin which the species is collected, and the location and age of the algalbody. As a result, alginates possessing a range of physiochemicalproperties, such as molecular weight and viscosity, are commerciallyavailable.

Alginates can be ionically crosslinked at room temperature and neutralpH to form hydrogels. The ability of alginates to form stable gels inphysiologically compatible conditions renders alginate gels useful in anumber of biomedical applications. For example, alginate gels have beused as a matrix for drug delivery to modulate the pharmacokinetics oftherapeutic, diagnostic, and prophylactic agents.

I. Definitions

“Alginate”, as used herein, is a collective term used to refer to linearpolysaccharides formed from β-D-mannuronate and α-L-guluronate in anyM/G ratio, as well as salts and derivatives thereof. The term“alginate”, as used herein, encompasses any polymer having the structureshown below, as well as salts thereof.

“Biocompatible”, as used herein, refers to a material which performs itsdesired function when introduced into an organism without inducingsignificant inflammatory response, immunogenicity, or cytotoxicity tonative cells, tissues, or organs. Biocompatibility, as used herein, canbe quantified using the in vivo biocompatibility assay described hereinin Example 5.

“Foreign Body Response”, as used herein, refers to the immunologicalresponse of biological tissue to the presence of any foreign material inthe tissue which can include protein adsorption, macrophages,multinucleated foreign body giant cells, fibroblasts, and angiogenesis.

“Chemically Modified Alginate” or “Modified Alginate”, are used hereininterchangeably, and refer to alginate polymers which contain one ormore covalently modified monomers.

“Covalently Modified Monomer”, as used herein, refers to a monomer whichis an analog or derivative of a mannuronate and/or guluronate monomerobtained from a mannuronate and/or guluronate monomer via a chemicalprocess.

“Contacting” as used herein in the context of coating refers to any wayfor coating a polymer, such as the modified alginate polymers disclosedherein, on a substrate or surface. Contacting can include, but is notlimited to, intraoperative dip-coating, spraying, wetting, immersing,dipping, painting, bonding or adhering, stepwise surface derivatization,or otherwise providing a substrate or surface with a compound with thehydrophobic, polycationic polymer. The polymer can be covalently ornon-covalently attached to the substrate or surface. In someembodiments, the polymer is non-covalently associated with the surface.

“Coating” as used herein refers to any temporary, semipermanent orpermanent layer, covering or surface. A coating can be applied as a gas,vapor, liquid, paste, semi-solid, or solid. In addition a coating can beapplied as a liquid and solidified into a hard coating. Elasticity canbe engineered into coatings to accommodate pliability, e.g. swelling orshrinkage, of the substrate or surface to be coated. Preferred coatingsare modified alginate polymers disclosed herein.

“Independently”, as used herein in the context of chemical formulae (andunless the context clearly indicates otherwise), means that eachinstance of the group referred to is chosen independently of the otherinstances of that group. For example, each instance of the group couldbe different from every other instance, some other instances, or noother instances of the group. Where multiple groups are referred to,“independently” means that each instance of each given group is chosenindependently of the other instances of the respective group and thateach of the groups are chosen independently of the other groups. Forexample, each instance of a first group could be different from everyinstance, some other instances, or no other instances of a second group(or third, or fourth, etc., group).

“Component” as used herein in the context of medical products, such asmedical devices, is a part of a product that is structurally integratedwith that product. A component may be applied to a substrate or to thesurface of a product, contained within the substance of the product,retained in the interior of the product, or any other arrangementwhereby that part is an integral element of the structure of theproduct. As an example, the silicone covering surrounding the mechanicalpart of a pacemaker is a component of the pacemaker. A component may bethe lumen of a product where the lumen performs some function essentialto the overall function of the product. The lumen of a tissue expanderport is a component of the tissue expander. A component can refer to areservoir or a discrete area within the product specifically adapted forthe delivery of a fluid to a surface of the product. A reservoir withinan implantable drug delivery device is a component of that device.

The phrase “effective amount,” as used herein in the context of acoating, generally refers to the amount of the coating applied to theimplant in order to provide one or more clinically measurable endpoints,such as reduced foreign body response compared to an uncoated implant,an implant coated with an unmodified coating, or another suitablecontrol. The phrase “effective amount,” as used herein in the context ofa cell, capsule, device, composition, or compound, refers to a nontoxicbut sufficient amount of the cell, capsule, device, composition, orcompound to provide the desired result. The exact amount required mayvary from subject to subject, depending on the species, age, and generalcondition of the subject; the severity of the disease that is beingtreated; the particular cell, capsule, device, composition, or compoundused; its mode of administration; and other routine variables. Anappropriate effective amount can be determined by one of ordinary skillin the art using only routine experimentation.

“Surface” or “surfaces,” as used herein, refers to any surface of anysolid or semi-solid material, including glass, plastics, metals,polymers, and like. This includes surfaces constructed out of more thanone material, including coated surfaces.

“Singularly Modified Alginate Polymer,” as used herein, refers tomodified alginates that contain one or more covalently modifiedmonomers, wherein substantially all of the covalently modified monomerspossess the same covalent modification (i.e. the polymer contains one‘type’ or species of covalently modified monomer). Singularly modifiedalginate polymers include, for example, modified alginate polymerswherein substantially all of the monomers in the modified alginatepolymer are represented by mannuronate monomers, guluronate monomers,and a covalently modified monomer defined by Formula I. Not all of themonomers are necessarily covalently modified.

“Capsule,” as used herein, refers to a particle having a mean diameterof about 150 μm to about 5 cm, formed of a cross-linked hydrogel, havinga cross-linked hydrogel core that is surrounded by one or more polymericshells, having one or more cross-linked hydrogel layers, having across-linked hydrogel coating, or a combination thereof. The capsule mayhave any shape suitable for, for example, cell encapsulation. Thecapsule may contain one or more cells dispersed in the cross-linkedhydrogel, thereby “encapsulating” the cells. Reference to “capsules”herein refers to and includes microcapsules unless the context clearlyindicates otherwise. Preferred capsules have a mean diameter of about150 μm to about 8 mm.

“Microcapsule” and “microgel,” as used herein, are used interchangeablyto refer to a particle or capsule having a mean diameter of about 150 μmto about 1000 μm.

“Biological material,” as used herein, refers to any biologicalsubstance, including, but not limited to, tissue, cells, biologicalmicromolecules, such as a nucleotides, amino acids, cofactors, andhormones, biological macromolecules, such as nucleic acids,polypeptides, proteins (for example enzymes, receptors, secretoryproteins, structural and signaling proteins, hormones, ligands, etc.),polysaccharides, and/or any combination thereof.

“Cell,” as used herein, refers to individual cells, cell lines, primarycultures, or cultures derived from such cells unless specificallyindicated. “Culture,” as used herein, refers to a composition includingisolated cells of the same or a different type. “Cell line,” as usedherein, refers to a permanently established cell culture that willproliferate indefinitely given appropriate fresh medium and space, thusmaking the cell line “immortal.” “Cell strain,” as used herein, refersto a cell culture having a plurality of cells adapted to culture, butwith finite division potential. “Cell culture,” as used herein, is apopulation of cells grown on a medium such as agar.

Cells can be, for example, xenogeneic, autologous, or allogeneic. Cellscan also be primary cells. Cells can also be cells derived from theculture and expansion of a cell obtained from a subject. For example,cells can also be stem cells or derived from stem cells. Cells can alsobe immortalized cells. Cells can also be genetically engineered toexpress a protein, nucleic acid, or other product.

“Mammalian cell,” as used herein, refers to any cell derived from amammalian subject.

“Autologous,” as used herein, refers to a transplanted biologicalmaterial, such as cells, taken from the same individual.

“Allogeneic,” as used herein, refers to a transplanted biologicalmaterial, such as cells, taken from a different individual of the samespecies.

“Xenogeneic,” as used herein, refers to a transplanted biologicalmaterial, such as cells, taken from a different species.

“Endocrine cell,” as used herein, refers to a cell of the endocrinesystem. “Secreting endocrine cell,” as used herein, refers to anendocrine cell that secretes one or more hormones.

“Islet cell,” as used herein, refers to an endocrine cell derived from amammalian pancreas. Islet cells include alpha cells that secreteglucagon, beta cells that secrete insulin and amylin, delta cells thatsecrete somatostatin, PP cells that secrete pancreatic polypeptide, orepsilon cells that secrete ghrelin. The term includes homogenous andheterogeneous populations of these cells. In preferred embodiments, apopulation of islet cells contains at least beta cells.

“Hormone-producing cell,” as used herein, refers to a cell that producesone or more hormones. Preferred hormone-producing cells produce hormonein response to physiological stimulus, such as the physiologicalstimulus that cause secretion of the hormone from an endocrine cell thatnaturally secretes the hormone. Secreting endocrine cells,hormone-producing cells derived from stem cells, and cells geneticallyengineered to produce hormone are examples of hormone-producing cells.

“Insulin-producing cell,” as used herein, refers to a cell that producesinsulin. Preferred insulin-producing cells produce insulin in responseto glucose levels. Islet beta cells, insulin-producing cells derivedfrom stem cells, and cells genetically engineered to produce insulin areexamples of insulin-producing cells.

“Transplant,” as used herein, refers to the transfer of a cell, tissue,or organ to a subject from another source. The term is not limited to aparticular mode of transfer. Encapsulated cells may be transplanted byany suitable method, such as by injection or surgical implantation.

“Primary cells,” “primary cell lines,” and “primary cultures,” as usedherein, are used interchangeably to refer to cells and cells culturesthat have been derived from a subject and allowed to grow in vitro for alimited number of passages, that is, splittings, of the culture.

“Mesenchymal stem cell” or “MSC,” as used herein, refer to multipotentstem cells present in or derived from mesenchymal tissue that candifferentiate into a variety of cell types, including: osteoblasts,chondrocytes, and adipocytes.

“Derived from,” as used herein, with respect to cells, refer to cellsobtained from tissue, cell lines, or cells, which are then cultured,passaged, differentiated, induced, etc., to produce the derived cells.For example, induced pluripotent stem cells are derived from somaticcells.

“Pluripotency,” as used herein, refers to the ability of cells todifferentiate into multiple types of cells in an organism. By“pluripotent stem cells,” it is meant cells that can self-renew anddifferentiate to produce all types of cells in an organism. By“multipotency” it is meant the ability of cells to differentiate intosome types of cells in an organism but not all, typically into cells ofa particular tissue or cell lineage.

“Multi-potent cells” and “adult stem cells,” as used herein, refer toany type of stem cell that is not derived from an embryo or fetus andgenerally has a limited capacity to generate new cell types (referred toas “multipotency”) and being committed to a particular lineage.

“Induced pluripotent stem cell,” as used herein, encompasses pluripotentstem cells, that, like embryonic stem (ES) cells, can be cultured over along period of time while maintaining the ability to differentiate intoall types of cells in an organism, but that, unlike ES cells (which arederived from the inner cell mass of blastocysts), are derived fromsomatic cells.

For clarity of discussion herein, singularly modified alginates aredefined using formulae illustrating the structure of the covalentlymodified monomers incorporated in the backbone and omitting themannuronate and guluronate monomers. For example, a singularly modifiedalginate polymer composed of mannuronate monomers, guluronate monomers,and a covalently modified monomer defined by Formula I, wherein X isNR₄, R₁ is methyl, and R₄, Y₁, and Y₂ are hydrogen, is illustratedherein by the structure below.

“Multiply Modified Alginate Polymer”, as used herein, refers to modifiedalginates that contain covalently modified monomers, whereinsubstantially all of the covalently modified monomers do not possess thesame covalent modification (i.e. the polymer contains two or moredifferent ‘types’ or species of covalently modified monomers). Multiplymodified alginate polymers include, for example, modified alginatepolymers wherein substantially all of the monomers in the modifiedalginate polymer are represented by mannuronate monomers, guluronatemonomers, and two or more different types of covalently modifiedmonomers defined by Formula I. As used in this context, a ‘type’ or‘species’ of covalently modified monomer refers to a covalent monomerdefined by Formula I, wherein all possible variable positions arechemically defined. Not all the monomers are covalently modified.

For clarity of discussion herein, modified alginates are defined usingformulae illustrating the covalently modified monomers incorporated inthe backbone and omitting the mannuronate and guluronate monomers. Forexample, a multiply modified alginate polymer composed of mannuronatemonomers, guluronate monomers, and two different types of covalentlymodified monomers, wherein the first type of covalently modified monomeris defined by Formula I, wherein X is NR₄, R₁ is methyl, and R₄, Y₁, andY₂ are hydrogen and the second type of covalently modified monomer isdefined by Formula I, wherein X is oxygen, R₁ is ethyl, and Y₁ and Y₂are hydrogen, is illustrated by the structure below.

“Analog” and “Derivative,” in the context of chemical compounds, areused herein interchangeably, and refer to a compound having a structuresimilar to that of a parent compound, but varying from the parentcompound by a difference in one or more certain components. Analogs orderivatives differ from the parent compound in one or more atoms,functional groups, or substructures, which are replaced with otheratoms, groups, or substructures. An analog or derivative can be imaginedto be formed, at least theoretically, from the parent compound via somechemical or physical process. The terms analog and derivative encompasscompounds which retain the same basic ring structure as the parentcompound, but possess one or more different substituents on the ring(s).For example, analog or derivative of mannuronate or guluronate refers tocompounds which retain the core of the monomer, e.g., the pyranose ring,but differ in or more substitutents on the ring.

“Mannuronate” and “Mannuronate Monomer”, as used herein, refer tomannuronic acid monomers as well as salts thereof.

“Guluronate” and “Guluronate Monomer”, as used herein, refer toguluronic acid monomers as well as salts thereof.

“Substantially”, as used herein, specifies an amount of 95% or more, 96%or more, 97% or more, 98% or more, or 99% or more.

“Glass Transition Temperature” (T_(g)), as used herein, refers to thetemperature at which a reversible transition is observed in amorphousmaterials from a hard and relatively brittle state into a molten orrubber-like state. T_(g) values for alginate polymers can beexperimentally determined using differential scanning calorimetry (DSC,heated and cooled at a rate of 10 K/min). In all cases herein, values ofT_(g) are measured using powder polymer samples.

“Click Chemistry”, as used herein, refers to chemical reactions used tocouple two compounds together which are high yielding, wide in scope,create only byproducts that can be removed without chromatography, arestereospecific, simple to perform, and can be conducted in easilyremovable or benign solvents. Examples of reactions which fulfill thesecriteria include the nucleophilic ring opening of epoxides andaziridines, non-aldol type carbonyl reactions, including the formationof hydrazones and heterocycles, additions to carbon-carbon multiplebonds, including Michael Additions, and cycloaddition reactions, such asa 1,3-dipolar cycloaddition reaction (i.e. a Huisgen cycloadditionreaction). See, for example, Moses, and Moorhouse, Chem Soc. Rev.36:1249-1262 (2007); Kolb and Sharpless, Drug Discovery Today.8(24):1128-1137 (2003); and Kolb et al., Angew. Chem. Int. Ed.40:2004-2021 (2001).

“Polyvalent Cation”, as used herein, refers to cations which have apositive charge greater than 1. Examples include, but are not limitedto, Ca²⁺, Ba²⁺, and Sr²⁺.

“Substituted”, as used herein, refers to all permissible substituents ofthe compounds or functional groups described herein. In the broadestsense, the permissible substituents include acyclic and cyclic, branchedand unbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, preferably 1-14 carbonatoms, and optionally include one or more heteroatoms such as oxygen,sulfur, or nitrogen grouping in linear, branched, or cyclic structuralformats. Representative substituents include alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, andpolypeptide groups.

Heteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. It is understood that“substitution” or “substituted” includes the implicit proviso that suchsubstitution is in accordance with permitted valence of the substitutedatom and the substituent, and that the substitution results in a stablecompound, i.e. a compound that does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

“Aryl”, as used herein, refers to C₅-C₁₀-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring systems. Broadly defined, “aryl”, as used herein,includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example, benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics”. The aromaticring can be substituted at one or more ring positions with one or moresubstituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (orquaternized amino), nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN; and combinations thereof.

“Aryl” further encompasses polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 41-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl”.

“Alkyl”, as used herein, refers to the radical of saturated orunsaturated aliphatic groups, including straight-chain alkyl, alkenyl,or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups,cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkylsubstituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, andcycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unlessotherwise indicated, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain,C₃-C₃₀ for branched chain), preferably 20 or fewer, more preferably 10or fewer, most preferably 6 or fewer. If the alkyl is unsaturated, thealkyl chain generally has from 2-30 carbons in the chain, preferablyfrom 2-20 carbons in the chain, more preferably from 2-10 carbons in thechain. Likewise, preferred cycloalkyls have from 3-20 carbon atoms intheir ring structure, preferably from 3-10 carbons atoms in their ringstructure, most preferably 5, 6 or 7 carbons in the ring structure.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

“Alkyl” includes one or more substitutions at one or more carbon atomsof the hydrocarbon radical as well as heteroalkyls. Suitablesubstituents include, but are not limited to, halogens, such asfluorine, chlorine, bromine, or iodine; hydroxyl; —NRR′, wherein R andR′ are independently hydrogen, alkyl, or aryl, and wherein the nitrogenatom is optionally quaternized; —SR, wherein R is hydrogen, alkyl, oraryl; —CN; —NO₂; —COOH; carboxylate; —COR, —COOR, or —CON(R)₂, wherein Ris hydrogen, alkyl, or aryl; azide, aralkyl, alkoxyl, imino,phosphonate, phosphinate, silyl, ether, sulfonyl, sulfonamido,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃; —CN;—NCOCOCH₂CH₂; —NCOCOCHCH; —NCS; and combinations thereof.

“Amino” and “Amine”, as used herein, are art-recognized and refer toboth substituted and unsubstituted amines, e.g., a moiety that can berepresented by the general formula:

wherein, R, R′, and R″ each independently represent a hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbonyl, —(CH₂)_(m)—R′″, or R and R′ taken together withthe N atom to which they are attached complete a heterocycle having from3 to 14 atoms in the ring structure; R′″ represents a hydroxy group,substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring,a cycloalkenyl ring, a heterocycle, or a polycycle; and m is zero or aninteger ranging from 1 to 8. In preferred embodiments, only one of R andR′ can be a carbonyl, e.g., R and R′ together with the nitrogen do notform an imide. In preferred embodiments, R and R′ (and optionally R″)each independently represent a hydrogen atom, substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, or—(CH₂)_(m)—R′″. Thus, the term ‘alkylamine’ as used herein refers to anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto (i.e. at least one of R, R′, or R″ is an alkylgroup).

“Carbonyl”, as used herein, is art-recognized and includes such moietiesas can be represented by the general formula:

wherein X is a bond, or represents an oxygen or a sulfur, and Rrepresents a hydrogen, a substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,—(CH₂)_(m)—R″, or a pharmaceutical acceptable salt, R′ represents ahydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, or—(CH₂)_(m)—R″; R″ represents a hydroxy group, substituted orunsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenylring, a heterocycle, or a polycycle; and m is zero or an integer rangingfrom 1 to 8. Where X is oxygen and R is defines as above, the moiety isalso referred to as a carboxyl group. When X is oxygen and R ishydrogen, the formula represents a ‘carboxylic acid’. Where X is oxygenand R′ is hydrogen, the formula represents a ‘formate’. In general,where the oxygen atom of the above formula is replaced by a sulfur atom,the formula represents a ‘thiocarbonyl’ group. Where X is sulfur and Ror R′ is not hydrogen, the formula represents a ‘thioester’. Where X issulfur and R is hydrogen, the formula represents a ‘thiocarboxylicacid’. Where X is sulfur and R′ is hydrogen, the formula represents a‘thioformate’. Where X is a bond and R is not hydrogen, the aboveformula represents a ‘ketone’. Where X is a bond and R is hydrogen, theabove formula represents an ‘aldehyde’.

“Heteroalkyl”, as used herein, refers to straight or branched chain, orcyclic carbon-containing radicals, or combinations thereof, containingat least one heteroatom. Suitable heteroatoms include, but are notlimited to, O, N, Si, P and S, wherein the nitrogen, phosphorous andsulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized.

Examples of saturated hydrocarbon radicals include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, andhomologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,n-octyl. Examples of unsaturated alkyl groups include, but are notlimited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, and3-butynyl.

“Alkoxy”, “alkylamino”, and “alkylthio” are used herein in theirconventional sense, and refer to those alkyl groups attached to theremainder of the molecule via an oxygen atom, an amino group, or asulfur atom, respectively.

“Alkylaryl”, as used herein, refers to an alkyl group substituted withan aryl group (e.g., an aromatic or hetero aromatic group).

“Heterocycle” or “heterocyclic”, as used herein, refers to a cyclicradical attached via a ring carbon or nitrogen of a monocyclic orbicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ringatoms, consisting of carbon and one to four heteroatoms each selectedfrom the group consisting of non-peroxide oxygen, sulfur, and N(Y)wherein Y is absent or is H, O, C₁-C₁₀ alkyl, phenyl or benzyl, andoptionally containing 1-3 double bonds and optionally substituted withone or more substituents. Examples of heterocyclic ring include, but arenot limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Heterocyclicgroups can optionally be substituted with one or more substituents asdefined above for alkyl and aryl.

“Halogen”, as used herein, refers to fluorine, chlorine, bromine, oriodine.

II. Modified Alginates

Described herein are alginate polymers that have been chemicallymodified to alter their biocompatibility and physical properties, aswell as methods of making thereof.

A. Structure of Modified Alginate Polymers

Modified alginates contain one or more covalently modified monomersdefined by Formula I

wherein,

X is oxygen, sulfur, or NR₄;

R₁ is hydrogen, or an organic grouping containing any number of carbonatoms, preferably 1-30 carbon atoms, more preferably 1-20 carbon atoms,more preferably 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative R₁ groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, sulfonyl,substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl,phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl,C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup;

Y₁ and Y₂ independently are hydrogen or —PO(OR₅)₂; or

Y₂ is absent, and Y₂, together with the two oxygen atoms to which Y₁ andY₂ are attached form a cyclic structure as shown in Formula II

wherein; and

R₂ and R₃ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, preferably 1-30 carbon atoms, morepreferably 1-20 carbon atoms, more preferably 1-14 carbon atoms, andoptionally including one or more heteroatoms such as oxygen, sulfur, ornitrogen grouping in linear, branched, or cyclic structural formats,representative R₂ and R₃ groupings being alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy,aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup; or

R₂ and R₃, together with the carbon atom to which they are attached,form a 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and

R₄ and R₅ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, preferably 1-30 carbon atoms, morepreferably 1-20 carbon atoms, more preferably 1-14 carbon atoms, andoptionally including one or more heteroatoms such as oxygen, sulfur, ornitrogen grouping in linear, branched, or cyclic structural formats,representative R₄ and R₅ groupings being alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy,aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup.

In some embodiments, the modified alginate polymer is a singularlymodified alginate polymer. In specific embodiments, the singularlymodified alginate polymer contains one or more covalently modifiedmonomers defined by Formula I, wherein R₁ includes an azide group, analkyne group, or a 1,2,3-triazole ring. In certain embodiments, thesingularly modified alginate polymer contains one or more covalentlymodified monomers defined by Formula I, wherein X is not oxygen and R₁is not an unsubstituted C₁-C₁₈ alkyl group, poly(ethylene glycol) chain,or cholesteryl moiety. In certain additional embodiments, the singularlymodified alginate polymer contains one or more covalently modifiedmonomers defined by Formula I, wherein X is not NR₄ and R₁ is not asubstituted or unsubstituted C₁-C₆ alkyl group, or a poly(ethyleneglycol) chain.

In alternative embodiments, the modified alginate polymer is a multiplymodified alginate polymer. In preferred embodiments, the multiplymodified alginate polymer possesses a polysaccharide backbone containingmannuronate monomers, guluronate monomers, a first species or type ofcovalently modified monomer defined by Formula I, and a second speciesor type of covalently modified monomer defined by Formula I. In otherembodiments, the multiply modified alginate polymer possesses apolysaccharide backbone containing mannuronate monomers, guluronatemonomers, and three or more different types of covalently modifiedmonomers defined by Formula I.

In some embodiments, the multiply modified alginate polymer contains twodifferent species of covalently modified monomers defined by Formula I,wherein in both species of monomer, X is NR₄. In other embodiments, themultiply modified alginate polymer contains two different species ofcovalently modified monomers defined by Formula I, wherein in bothspecies of monomer, X is oxygen. In further embodiments, the multiplymodified alginate polymer contains two different species of covalentlymodified monomers defined by Formula I, wherein in one species ofmonomer X is oxygen, and in the second species of monomer, X is NR₄.

In some embodiments, the multiply modified alginate polymer contains twodifferent species of covalently modified monomers defined by Formula I,wherein in at least one species of monomer, R₁ includes one or morecyclic moieties. In preferred embodiments, the multiply modifiedalginate polymer contains two different species of covalently modifiedmonomers defined by Formula I, wherein in at least one species ofmonomer, R₁ includes a phenyl ring, furan ring, oxolane ring, dioxolanering, or a 1,2,3-triazole ring.

In certain embodiments, the multiply modified alginate polymer containstwo different species of covalently modified monomers defined by FormulaI, wherein in at least one species of monomer, R₁ includes one or morehalogen moieties, an azide group, or an alkyne.

In preferred embodiments, the multiply modified alginate polymer is oneof the multiply modified alginate polymers shown below.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy,C₁-C₁₀ alkylamino, C₁-C₁₀ alkylthio, C₁-C₉ alkyl, C₁-C₉ alkoxy, C₁-C₉alkylamino, C₁-C₉ alkylthio, C₁-C₈ alkyl, C₁-C₈ alkoxy, C₁-C₈alkylamino, C₁-C₈ alkylthio, C₁-C₇ alkyl, C₁-C₇ alkoxy, C₁-C₇alkylamino, C₁-C₇ alkylthio, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylamino, C₁-C₆ alkylthio, C₁-C₅ alkyl, C₁-C₅ alkoxy, C₁-C₅alkylamino, C₁-C₅ alkylthio, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄alkylamino, C₁-C₄ alkylthio, C₁-C₃ alkyl, C₁-C₃ alkoxy, C₁-C₃alkylamino, C₁-C₃ alkylthio, C₁-C₂ alkyl, C₁-C₂ alkoxy, C₁-C₂alkylamino, C₁-C₂ alkylthio, C₁₀ alkyl, C₁₀ alkoxy, C₁₀ alkylamino, C₁₀alkylthio, C₉ alkyl, C₉ alkoxy, C₉ alkylamino, C₉ alkylthio, C₈ alkyl,C₈ alkoxy, C₈ alkylamino, C₈ alkylthio, C₇ alkyl, C₇ alkoxy, C₇alkylamino, C₇ alkylthio, C₆ alkyl, C₆ alkoxy, C₆ alkylamino, C₆alkylthio, C₈ alkyl, C₅ alkoxy, C₅ alkylamino, C₅ alkylthio, C₄ alkyl,C₄ alkoxy, C₄ alkylamino, C₄ alkylthio, C₃ alkyl, C₃ alkoxy, C₃alkylamino, C₃ alkylthio, C₂ alkyl, C₂ alkoxy, C₂ alkylamino, C₂alkylthio, C₁ alkyl, C₁ alkoxy, C₁ alkylamino, or C₁ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy,C₁-C₁₀ alkylamino, or C₁-C₁₀ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁-C₉ alkyl, C₁-C₉ alkoxy,C₁-C₉ alkylamino, or C₁-C₉ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁-C₈ alkyl, C₁-C₈ alkoxy,C₁-C₈ alkylamino, or C₁-C₈ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁-C₇ alkyl, C₁-C₇ alkoxy,C₁-C₇ alkylamino, or C₁-C₇ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkoxy,C₁-C₆ alkylamino, or C₁-C₆ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), and R^(d) are independently hydrogen, amino, hydroxyl, thiol,oxo, or substituted or unsubstituted C₁-C₅ alkyl, C₁-C₅ alkoxy, C₁-C₅alkylamino, or C₁-C₅ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁-C₄ alkyl, C₁-C₄ alkoxy,C₁-C₄ alkylamino, or C₁-C₄ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁-C₃ alkyl, C₁-C₃ alkoxy,C₁-C₃ alkylamino, or C₁-C₃ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁-C₂ alkyl, C₁-C₂ alkoxy,C₁-C₂ alkylamino, or C₁-C₂ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁₀ alkyl, C₁₀ alkoxy, C₁₀alkylamino, or C₁₀ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₉ alkyl, C₉ alkoxy, C₉alkylamino, or C₉ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₈ alkyl, C₈ alkoxy, C₈alkylamino, or C₈ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₇ alkyl, C₇ alkoxy, C₇alkylamino, or C₇ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₆ alkyl, C₆ alkoxy, C₆alkylamino, or C₆ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₅ alkyl, C₅ alkoxy, C₅alkylamino, or C₅ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₄ alkyl, C₄ alkoxy, C₄alkylamino, or C₄ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₃ alkyl, C₃ alkoxy, C₃alkylamino, or C₃ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₂ alkyl, C₂ alkoxy, C₂alkylamino, or C₂ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently hydrogen, amino, hydroxyl,thiol, oxo, or substituted or unsubstituted C₁ alkyl, C₁ alkoxy, C₁alkylamino, or C₁ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, C₁-C₁₀alkylamino, C₁-C₁₀ alkylthio, C₁-C₉ alkyl, C₁-C₉ alkoxy, C₁-C₉alkylamino, C₁-C₉ alkylthio, C₁-C₈ alkyl, C₁-C₈ alkoxy, C₁-C₈alkylamino, C₁-C₈ alkylthio, C₁-C₇ alkyl, C₁-C₇ alkoxy, C₁-C₇alkylamino, C₁-C₇ alkylthio, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylamino, C₁-C₆ alkylthio, C₁-C₅ alkyl, C₁-C₅ alkoxy, C₁-C₅alkylamino, C₁-C₅ alkylthio, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄alkylamino, C₁-C₄ alkylthio, C₁-C₃ alkyl, C₁-C₃ alkoxy, C₁-C₃alkylamino, C₁-C₃ alkylthio, C₁-C₂ alkyl, C₁-C₂ alkoxy, C₁-C₂alkylamino, C₁-C₂ alkylthio, C₁₀ alkyl, C₁₀ alkoxy, C₁₀ alkylamino, C₁₀alkylthio, C₉ alkyl, C₉ alkoxy, C₉ alkylamino, C₉ alkylthio, C₈ alkyl,C₈ alkoxy, C₈ alkylamino, C₈ alkylthio, C₇ alkyl, C₇ alkoxy, C₇alkylamino, C₇ alkylthio, C₆ alkyl, C₆ alkoxy, C₆ alkylamino, C₆alkylthio, C₅ alkyl, C₅ alkoxy, C₅ alkylamino, C₅ alkylthio, C₄ alkyl,C₄ alkoxy, C₄ alkylamino, C₄ alkylthio, C₃ alkyl, C₃ alkoxy, C₃alkylamino, C₃ alkylthio, C₂ alkyl, C₂ alkoxy, C₂ alkylamino, C₂alkylthio, C₁ alkyl, C₁ alkoxy, C₁ alkylamino, or C₁ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, C₁-C₁₀alkylamino, or C₁-C₁₀ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₉ alkyl, C₁-C₉ alkoxy, C₁-C₉alkylamino, or C₁-C₉ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₈ alkyl, C₁-C₈ alkoxy, C₁-C₈alkylamino, or C₁-C₈ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₇ alkyl, C₁-C₇ alkoxy, C₁-C₇alkylamino, or C₁-C₇ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆alkylamino, or C₁-C₆ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₅ alkyl, C₁-C₅ alkoxy, C₁-C₅alkylamino, or C₁-C₅ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄alkylamino, or C₁-C₄ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₃ alkyl, C₁-C₃ alkoxy, C₁-C₃alkylamino, or C₁-C₃ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁-C₂ alkyl, C₁-C₂ alkoxy, C₁-C₂alkylamino, or C₁-C₂ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently amino, hydroxyl, thiol, oxo, orsubstituted or unsubstituted C₁₀ alkyl, C₁₀ alkoxy, C₁₀ alkylamino, orC₁₀ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₉ alkyl, C₉ alkoxy, C₉ alkylamino, orC₉ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₈ alkyl, C₈ alkoxy, C₈ alkylamino, orC₈ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₇ alkyl, C₇ alkoxy, C₇ alkylamino, orC₇ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₆ alkyl, C₆ alkoxy, C₆ alkylamino, orC₆ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₅ alkyl, C₅ alkoxy, C₅ alkylamino, orC₅ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₄ alkyl, C₄ alkoxy, C₄ alkylamino, orC₄ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d) and R^(e) are independently amino, hydroxyl, thiol, oxo, orsubstituted or unsubstituted C₃ alkyl, C₃ alkoxy, C₃ alkylamino, or C₃alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₂ alkyl, C₂ alkoxy, C₂ alkylamino, orC₂ alkylthio.

In some embodiments, R₁ through R₁₇, R₃₁ through R₃₆, R^(a), R^(b),R^(c), R^(d), and R^(e) are independently amino, hydroxyl, thiol, oxo,or substituted or unsubstituted C₁ alkyl, C₁ alkoxy, C₁ alkylamino, orC₁ alkylthio.

Modified alginate polymers can be of any desired molecular weight. Theweight average molecular weight of the alginates is preferably between1,000 and 1,000,000 Daltons, more preferably between 10,000 and 500,000Daltons as determined by gel permeation chromatography.

Modified alginate polymers can contain any ratio of mannuronatemonomers, guluronate monomers, and covalently modified monomers. In someembodiments, greater than 2.5%, 5%, 7.5%, 10%, 12%, 14%, 15%, 16%, 18%,20%, 22%, 24%, 25%, 26%, 28%, 30%, 32.5%, 35%, 37.5%, 40%, 45%, 50%,55%, or 60% of the monomers in the modified alginate polymer arecovalently modified monomers. Preferably greater than 10%, morepreferably greater than 20%, and most preferably greater than 30% of themonomers in the modified alginate polymer are covalently modifiedmonomers.

Modified alginate polymers can be produced incorporating covalentlymodified monomers possessing a range of different hydrogen bondingpotentials, hydrophobicities/hydrophilicities, and charge states. Theinclusion of covalently modified monomers into an alginate polymeralters the physiochemical properties of alginate polymer. Accordingly,the physiochemical properties of alginates can be tuned for desiredapplications by the selective incorporation of covalently modifiedmonomers.

For example, the glass transition temperature (T_(g)), can be varied bythe incorporation of covalently modified monomers. In some embodiments,the modified alginate polymer powder possess a T_(g), as measured bydifferential scanning calorimetry (DSC), of greater than 50° C., 60° C.,65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., 105°C., 110° C., 115° C., 120° C., 125° C., 130° C., 135° C., 140° C., 145°C., 150° C., 160° C., 175° C., 190° C., or 200° C.

The hydrophobicity/hydrophilicity of alginates can be varied by theincorporation of hydrophobic and/or hydrophilic covalently modifiedmonomers. In preferred embodiments, the modified alginate polymercontains one or more hydrophobic covalently modified monomers. Therelative hydrophobicity/hydrophilicity of modified alginates can bequantitatively assessed by measuring the contact angle of a waterdroplet on a film of the modified alginate polymer using a goniometer.In some embodiments, the modified alginate has a contact angle of lessthan 90° (i.e. it is hydrophilic). In preferred embodiments, themodified alginate has a contact angle of more than 90° (i.e. it ishydrophobic). In some embodiments, the modified alginate has a contactangle of more than 95°, 100°, 105°, 110°, 115°, or 120°.

In embodiments used for cell encapsulation, the modified alginatepolymer can be ionically crosslinked by a polyvalent cation such asCa²⁺, Sr²⁺, or Ba²⁺ to form hydrogels. The ability of modified alginatesto form stable hydrogels in physiological conditions can be quantifiedusing the hydrogel formation assay described in Example 2.

In some embodiments, the modified alginate polymer forms hydrogels suchthat the fluorescence intensity measured using the high throughputhydrogel formation assay described herein is greater than 10,000,15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or55,000. In preferred embodiments, the modified alginate polymer formshydrogels such that the fluorescence intensity measured using the highthroughput hydrogel formation assay described herein is greater than15,000. In preferred embodiments, the modified alginate polymer formshydrogels such that the fluorescence intensity measured using the highthroughput hydrogel formation assay described herein is between 15,000and 55,000, preferably between 20,000 and 55,000, more preferablybetween 25,000 and 55,000.

In embodiments used for cell encapsulation, the modified alginatepolymer forms a hydrogel with sufficient porosity to permit nutrients,waste, and the hormones and/or proteins secreted from encapsulated cellsto diffuse freely into and out of the capsules, while simultaneouslypreventing the incursion of immune cells into the gel matrix. Theporosity and surface area of modified alginate hydrogels can be measuredusing BET analysis. Prior to BET analysis, solvent and volatileimpurities are removed by prolonged heating of the modified alginate gelunder vacuum. Subsequently, the hydrogel samples are cooled undervacuum, for example by liquid nitrogen, and analyzed by measuring thevolume of gas (typically N₂, Kr, CO₂, or Ar gas) adsorbed to thehydrogel at specific pressures. Analysis of the physisorption of the gasat variable pressures is used to characterize the total surface area andporosity of gels formed by the modified alginate polymers. The preferredmethod of determining hydrogel porosity is BET analysis.

In preferred embodiments, the modified alginate forms a hydrogel withsufficient porosity to permit nutrients, waste, and the hormones and/orproteins secreted from encapsulated cells to diffuse freely into and outof the capsules, while simultaneously preventing the incursion of immunecells into the gel matrix. In some embodiments, the porosity of thehydrogel formed by the modified alginate polymer is increased by 5%,10%, 15%, or 20% relative to the porosity of a hydrogel formed from theunmodified alginate polymer. In alternative embodiments, the porosity ofthe hydrogel formed by the modified alginate polymer is decreased by 5%,10%, 15%, or 20% relative to the porosity of a hydrogel formed from theunmodified alginate polymer.

In preferred embodiments used for cell encapsulation, the modifiedalginate is biocompatible. The biocompatibility of modified alginatescan be quantitatively determined using the fluorescence-based in vivobiocompatibility assay described in Example 5. In this assay, cathepsinactivity was measured using an in vivo fluorescence assay to quantifythe foreign body response to the modified alginate.

In some embodiments, the modified alginate polymer is biocompatible suchthat the fluorescence response normalized to unmodified alginatemeasured using the in vivo biocompatibility assay described herein isless than 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,or 40%. In preferred embodiments, the modified alginate polymer inducesa lower foreign body response than unmodified alginate. This isindicated by fluorescence response normalized to unmodified alginate ofless than 100%. In some embodiments, the modified alginate polymer isbiocompatible such that the fluorescence response normalized tounmodified alginate measured using the in vivo biocompatibility assaydescribed herein is less than 75%, more preferably less than 65%, andmost preferably less than 50%.

B. Capsules and Particle Morphology

Capsules are particles having a mean diameter of about 150 μm to about 5cm. The disclosed capsules can be formed of cross-linked hydrogel. Otherthan the encapsulated material, the capsules, for example, can be formedsolely of cross-linked hydrogel, can have a cross-linked hydrogel corethat is surrounded by one or more polymeric shells, can have one or morecross-linked hydrogel layers, can have a cross-linked hydrogel coating,or a combination thereof. The capsule may have any shape suitable for,for example, cell encapsulation. The capsule may contain one or morecells dispersed in the cross-linked hydrogel, thereby “encapsulating”the cells. Preferred capsules are formed of or include one or more ofthe disclosed modified alginates. Preferred capsules have a meandiameter of about 150 μm to about 8 mm.

The capsules can have any mean diameter from about 150 μm to about 5 cm.Preferably the capsules have a mean diameter that is greater than 1 mm,preferably 1.5 mm or greater. In some embodiments, the capsules can beas large as about 8 mm in diameter. For example, the capsule can be in asize range of about 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mmto 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm, 1.5 mm to 3 mm, or 1.5 mm to 2mm.

The rate of molecules entering the capsule necessary for cell viabilityand the rate of therapeutic products and waste material exiting thecapsule membrane can be selected by modulating capsule permeability.Capsule permeability can also be modified to limit entry of immunecells, antibodies, and cytokines into the capsule. Generally, as shownby the examples, known methods of forming hydrogel capsules can producecapsules the permeability of which limit entry of immune cells,antibodies, and cytokines into the capsule. Since different cell typeshave different metabolic requirements, the permeability of the membranecan be optimized based on the cell type encapsulated in the hydrogel.The diameter of the capsules is an important factor that influences boththe immune response towards the cell capsules as well as the masstransport across the capsule membrane.

The growing recognition of the parameters driving fibrosis in vivo hasbeen applied to the analysis of the performance of modified alginates.Intraperitoneal (IP) implantation of modified alginate capsules revealedthat modified alginates may result in abnormally shaped capsules whencrosslinked using conditions defined for unmodified alginates. Theseabnormally shaped capsules can complicate implementation andinterpretation of modified alginate capsules implanted IP. In an effortto improve the capsule morphology, formulation methods for use withmodified alginate microparticles were developed where modified alginateswere blended with a small amount of high molecular weight alginate.Particles prepared from this mixture yielded particles with improvedmorphology and stability.

The unmodified alginate typically has a weight average molecular weightof about 50,000 Daltons to about 500,000 Daltons; however, unmodifiedalginates having molecular weights can also be used. In someembodiments, the weight average molecular weight is from about 50,000 toabout 250,000 Daltons, more preferably from about 50,000 to about150,000 Daltons. In some embodiments, the weight average molecularweight is about 100,000 Daltons.

In other embodiments, one or more additional hydrogel-forming polymersare used in combination with unmodified alginate or in place ofunmodified alginate. Such polymers are known in the art. Examplesinclude, but are not limited to, PEG, chitosan, dextran, hyaluronicacid, silk, fibrin, poly(vinyl alcohol) and poly(hydroxyl ethylmethacrylate).

For example, particles prepared from modified alginate 263_A12microparticles formulated with barium and mannitol were compared toparticles prepared from 263_A12 blended with a small amount ofunmodified SLG100 alginate (16% by weight). The particles prepared froma mixture of modified alginate and unmodified alginate produced morehomogenous microparticle populations in terms of shape and size asevaluated by scanning electron microscopy (SEM). Quantitativefluorescence analysis with prosense at several time points with modifiedalginates blended with SLG100 showed that several reformulated modifiedalginates display less inflammatory response at day 7 compared to thecontrol alginate. Initial experiments with large capsules (1.5 mmdiameter) were comparably clean capsules after 2 weeks in the IP spaceof immunocompetent C57BL6 mice. Subsequent experiments (Example 9) showthat encapsulated human cells can achieve glucose-responsive, long-termglycemic correction (over 170 days) in an immune-competent diabeticanimal with no immunosuppression. This result was accomplished using amodified alginate as disclosed to encapsulate the human cells. Theresulting capsule mitigates immunological responses to human cellimplants, effectively delaying the fibrotic deposition that leads toimplant tissue necrosis. This formulation provided sufficientimmunoprotection to enable long-term glycemic correction, in spite ofthe xenogeneic stimulation that these human cells manifest in animmunocompetent rodent recipient.

Because the disclosed modified alginates mediate the reduced fibrosis,capsules made of other materials but coated or encapsulated with themodified alginates is a useful form of capsule for achieving reducedfibrosis. This, the capsules can include capsules and particles made ofa variety of materials that are then coated or encapsulated in alginatethat is or included modified alginate.

The disclosed compositions may be fabricated into artificial organs,such as an artificial pancreas containing encapsulated islet cells. Insome of these embodiments, the cells are encapsulated in a singlehydrogel compartment. In other embodiments, the composition contains aplurality of encapsulated cells dispersed or encapsulated in abiocompatible structure.

C. Preparation of Modified Alginate Polymers

Modified alginates can be prepared through covalent modification of anyavailable alginate polymer. Covalently modified monomers can beintroduced into alginate polymers using a variety of syntheticprocedures known in the art.

In some embodiments, mannuronate and guluronate monomers are covalentlymodified via esterification and/or amidation of their carboxylic acidmoiety. In alternative embodiments, mannuronate and guluronate monomersare covalently modified via phosphorylation or acetal formation.Stoichiometric variation of the reactants during covalent modificationcan be used to vary the amount of covalently modified monomerincorporated into the modified alginate.

In addition to the reactions discussed below, alternative syntheticmethodologies for the covalent modification of mannuronate andguluronate monomers are known in the art. (see, for example, March,“Advanced Organic Chemistry,” 5^(th) Edition, 2001, Wiley-IntersciencePublication, New York).

1. Modification Via the Carboxylate Moiety of the Mannuronate andGuluronate Monomers

Mannuronate and guluronate monomers contain a carboxylic acid moietywhich can serve as a point of covalent modification. In preferredembodiments, the carboxylic acid moiety present on one or moremannuronate and/or guluronate residues (1) are reacted as shown inScheme 1.

Mannuronate and guluronate residues (A) can be readily esterified by avariety of methods known in the art, forming covalently modified monomerB. For example, using a Steglich Esterification, mannuronate andguluronate residues (A) can be esterified by reaction with any suitablealcohol (HO—R₁) in the presence of a carbodiimide (for example,N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC),or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)) anddimethylaminopyridine (DMAP). In a preferred method, mannuronate andguluronate residues (A) were esterified by reaction with a large molarexcess of an alcohol (HO—R₁) in the presence of2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methyl morpholine(NMM). See, for example, Garrett, C. E. et al. Tetrahedron Lett. 2002;43(23): 4161-4164. Preferred alcohols for use as reagents inesterification include those shown below.

Mannuronate and guluronate residues (A) can also be covalently modifiedvia amidation, forming modified monomer C. For example, mannuronate andguluronate residues (A) can amidated by reaction with any suitable amine(R₁—NH₂) in the presence of a carbodiimide and DMAP. In a preferredmethod, mannuronate and guluronate residues (A) were amidated byreaction with a stoichiometric amount of a suitable amine (R₁—NH₂) inthe presence of CDMT and NMM. Preferred amines for use as reagents inamidation reactions include those shown below.

2. Modification of Mannuronate and Guluronate Monomers Via ClickChemistry

In some embodiments, mannuronate and guluronate monomers are covalentlymodified to introduce a functional group which can be further reactedvia click chemistry.

In preferred embodiments, amidation and/or esterification is used tointroduce a functional group which can further reacted using a1,3-dipolar cycloaddition reaction (i.e. a Huisgen cycloadditionreaction). In a 1,3-dipolar cycloaddition reaction, a first moleculecontaining an azide moiety is reacted with a second molecule containinga terminal or internal alkyne. As shown below, the azide and the alkynegroups undergo an intramolecular 1,3-dipolar cycloaddition reaction,coupling the two molecules together and forming a 1,2,3-triazole ring.

The regiochemistry of 1,3-dipolar cycloadditions reaction can becontrolled by addition of a copper(I) catalyst (formed in situ by thereduction of CuSO₄ with sodium ascorbate) or a ruthenium catalyst (suchas Cp*RuCl(PPh₃)₂, Cp*Ru(COD), or Cp*[RuCl₄]). For example, using acopper catalyst, azides and terminal alkynes can be reacted toexclusively afford the 1,4-regioisomers of 1,2,3-triazoles. Similarly,in the presence of a suitable ruthenium catalyst, azides can be reactedwith internal or terminal alkynes to form exclusively the1,5-regioisomers of 1,2,3-triazoles.

In some embodiments, amidation and/or esterification is used to form acovalently modified monomer containing an alkyne moiety. In theseembodiments, the alkyne moiety present on the covalently modifiedmonomer can be further reacted with a second molecule containing anazide functional group. Upon reaction, the azide and the alkyne groupsundergo an intramolecular 1,3-dipolar cycloaddition reaction forming a1,2,3-triazole ring, coupling the second molecule to the covalentlymodified monomer.

Examples of the alkyne-containing amidation/esterification reactantinclude X_(a)—R_(z)—C≡C—R_(x); wherein X_(a) is —OH or —NH₂; whereinR_(z) is alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group; and wherein R_(x) is hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, sulfonyl,substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl,phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl,C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup.

In some embodiments,

(1) R_(z) is hydrogen,

wherein y is an integer from 1 to 11; wherein R^(e) is independentlyalkoxy, amino, alkylamino, dialkylamino, hydroxy, alkenyl, alkynyl,substituted alkyl, substituted alkenyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, and polypeptidegroup; or together with the carbon atom to which they are attached, forma 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; wherein one instance of R^(e) is or contains X_(a);wherein R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, and R₂₃ are independently C, O, N, orS, wherein the bonds between adjacent R₁₈ to R₂₃ are double or singleaccording to valency, and wherein R₁₈ to R₂₃ are bound to none, one, ortwo hydrogens according to valency; and wherein R₂₄ is independently—(CR₂₅R₂₅)_(p)—or —(CR₂₅R₂₅)_(p)—X_(b)—(CR₂₅R₂₅)_(q)—, wherein p and qare independently integers from 0 to 5, wherein X_(b) is absent, —O—,—S—, —SO₂—, or NR₄, wherein each R₂₅ is independently absent, hydrogen,═O, ═S, —OH, —SH, —NR₄, wherein R₄ is alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup;

(B) —(CH₂)₈—R₂₆, wherein s is an integer from 0 to 20; wherein R₂₆ is—X_(a), —O—R₂₇, —S—R₂₇, —(CH₂)_(r)—R₂₇, —CO—R₂₇, or —CHR₂₈R₂₉, wherein ris an integer from 0 to 19; wherein R₂₇ is —X_(a), —(CH₂)_(u)—R₃₀,wherein u is an integer from 0 to 18; wherein R₂₈ is —(CH₂)_(t)—R₃₀, R₂₉is —(CH₂)_(v)—R₃₀, and t and v are integers from 0 to 18, wherein t andv together total 0 to 18; wherein R₃₀ is —X_(a), methyl, —OH, —SH, or—COOH; or

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group; and wherein one instance of R₃₁,R₃₂, R₃₃, R₃₄, R₃₅, or R₃₆ is or contains X_(a);(2) R_(x) is hydrogen,

wherein y is an integer from 0-11; wherein R^(e) is independentlyalkoxy, amino, alkylamino, dialkylamino, hydroxy, alkenyl, alkynyl,substituted alkyl, substituted alkenyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, and polypeptidegroup; or together with the carbon atom to which they are attached, forma 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; wherein R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, and R₂₃ areindependently C, O, N, or S, wherein the bonds between adjacent R₁₈ toR₂₃ are double or single according to valency, and wherein R₁₈ to R₂₃are bound to none, one, or two hydrogens according to valency; andwherein R₂₄ is independently —(CR₂₅R₂₅)_(p)— or—(CR₂₅R₂₅)_(p)—X_(b)—(CR₂₅R₂₅)_(q)—, wherein p and q are independentlyintegers from 0 to 5, wherein X_(b) is absent, —O—, —S—, —SO₂—, or NR₄,wherein each R₂₅ is independently absent, hydrogen, ═O, ═S, —OH, —SH,—NR₄, wherein R₄ is alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group;

(B) —(CH₂)₈—R₂₆, wherein s is an integer from 0 to 20; wherein R₂₆ is—O—R₂₇, —S—R₂₇, —(CH₂)_(r)—R₇, —CO—R₂₇, —CO—R₂₇, or —CHR₂₈R₂₉, wherein ris an integer from 0 to 19; wherein R₂₇ is —(CH₂)_(u)—R₃₀, wherein u isan integer from 0 to 18; wherein R₂₈ is —(CH₂)_(t)—R₃₀, R₂₉ is—(CH₂)_(v)—R₃₀, and t and v are integers from 0 to 18, wherein t and vtogether total 0 to 18; wherein R₃₀ is methyl, —OH, —SH, or —COOH; or

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group; and(3) wherein R_(z) and R_(x) are not both hydrogen.

Examples of the azide-containing second molecule include R_(w)—N₃,wherein R_(w) is alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, R_(w) is

wherein k are independently an integer from 1 to 30; wherein z is aninteger from 0 to 4; wherein X_(d) is O or S; wherein R^(a) isindependently alkoxy, amino, alkylamino, dialkylamino, hydroxy, alkenyl,alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl,phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group; or together with the carbon atom to which they areattached, form a 3- to 8-membered unsubstituted or substitutedcarbocyclic, or heterocyclic ring; and wherein R₁₀, R₁₁, R₁₂, R₁₃, R₁₄,R₁₅, R₁₆, R₁₇ are independently hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy,aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, sulfonyl, substituted sulfonyl,sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In alternative embodiments, amidation and/or esterification is used toform a covalently modified monomer containing an azide moiety. In theseembodiments, the azide moiety present on the covalently modified monomercan be further reacted with a second molecule containing a terminal orinternal alkyne. Upon reaction, the azide and the alkyne groups undergoan intramolecular 1,3-dipolar cycloaddition reaction forming a1,2,3-triazole ring, coupling the second molecule to the covalentlymodified monomer.

Examples of the azide-containing amidation/esterification reactantinclude X_(c)—R_(w)—N₃, where X_(c) is —OH or —NH₂ and R_(w) is alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, sulfonyl, substitutedsulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group.

In some embodiments, X_(c) is not —NH₂ and R_(w) is not —CH₂—Ar— or—CH₂—CH₂—(O—CH₂—CH₂)₃—.

In some embodiments, R_(w) is

wherein k are independently an integer from 1 to 30; wherein z is aninteger from 0 to 4; wherein X_(d) is O or S; wherein R^(a) isindependently alkoxy, amino, alkylamino, dialkylamino, hydroxy, alkenyl,alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl,phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group; or together with the carbon atom to which they areattached, form a 3- to 8-membered unsubstituted or substitutedcarbocyclic, or heterocyclic ring; wherein one instance of R^(a) is orcontains X_(c); wherein R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇ areindependently hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group; and wherein one instance of R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, or R₁₇ is or contains X_(c); or

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group; and wherein one instance of R₃₁,R₃₂, R₃₃, R₃₄, R₃₅, or R₃₆ is or contains X_(c).

Examples of the alkyne-containing second molecule includeR_(z)—C≡C—R_(x), wherein R_(z) and R_(x) are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, sulfonyl,substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl,phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl,C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup.

In some embodiments, R_(z) and R_(x) are independently hydrogen,

wherein y is an integer from 0 to 11; wherein R^(e) is independentlyalkoxy, amino, alkylamino, dialkylamino, hydroxy, alkenyl, alkynyl,substituted alkyl, substituted alkenyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, and polypeptidegroup; or together with the carbon atom to which they are attached, forma 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; wherein R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, and R₂₃ areindependently C, O, N, or S, wherein the bonds between adjacent R₁₈ toR₂₃ are double or single according to valency, and wherein R₁₈ to R₂₃are bound to none, one, or two hydrogens according to valency; andwherein R₂₄ is independently —(CR₂₅R₂₅)_(p)— or—(CR₂₅R₂₅)_(p)—X_(b)—(CR₂₅R₂₅)_(q)—, wherein p and q are independentlyintegers from 0 to 5, wherein X_(b) is absent, —O—, —S—, —SO₂—, or NR₄,wherein each R₂₅ is independently absent, hydrogen, ═O, ═S, —OH, —SH,—NR₄, wherein R₄ is alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group;

(B) —(CH₂)_(s)—R₂₆, wherein s is an integer from 0 to 20; wherein R₂₆ is—O—R₂₇, —S—R₂₇, —(CH₂)_(r)—R₂₇, —CO—R₂₇, or —CHR₂₈R₂₉, wherein r is aninteger from 0 to 19; wherein R₂₇ is —(CH₂)_(u)—R₃₀, wherein u is aninteger from 0 to 18; wherein R₂₈ is —(CH₂)_(t)—R₃₀, R₂₉ is—(CH₂)_(v)—R₃₀, and t and v are integers from 0 to 18, wherein t and vtogether total 0 to 18; wherein R₃₀ is methyl, —OH, —SH, or —COOH; or

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group; and

wherein R_(z) and R_(x) are not both hydrogen.

In some embodiments, the azide moiety can be added to a covalentlymodified monomer containing a leaving group, such as I, Br, OTs, OMs. Insome embodiments, amidation and/or esterification is used to form thecovalently modified monomer containing the leaving group. Examples ofthe leaving group-containing amidation/esterification reactant includeX_(c)—R_(w)-L, where X_(c) is —OH or —NH₂, L is the leaving group, andR_(w) is alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group.

In some embodiments, X_(c) is not —NH₂ and R_(w) is not —CH₂—Ar— or—CH₂—CH₂—(O—CH₂—CH₂)₃—.

In some embodiments, R_(w) is

wherein k are independently an integer from 1 to 30; wherein z is aninteger from 0 to 4; wherein X_(d) is O or S; wherein R^(a) isindependently alkoxy, amino, alkylamino, dialkylamino, hydroxy, alkenyl,alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl,phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group; or together with the carbon atom to which they areattached, form a 3- to 8-membered unsubstituted or substitutedcarbocyclic, or heterocyclic ring; wherein one instance of R^(a) is orcontains X_(c); wherein R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇ areindependently hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group; and wherein one instance of R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, or R₁₇ is or contains X_(c); or

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, aminoacid, poly(ethyleneglycol), peptide, or polypeptide group; and wherein one instance of R₃₁,R₃₂, R₃₃, R₃₄, R₃₅, or R₃₆ is or contains X_(c).

In some embodiments, X_(c) is not —NH₂ and R_(w) is not —CH₂—Ar— or—CH₂—CH₂—(O—CH₂—CH₂)₃—.

In preferred embodiments, amidation is used to form a covalentlymodified monomer containing an azide moiety. Subsequently, the azidemoiety present on the covalently modified monomer is reacted with asecond molecule containing a terminal or internal alkyne, forming a1,2,3-triazole ring and coupling the second molecule to the covalentlymodified monomer.

As shown in Scheme 2, different strategies can be employed to preparecovalently modified monomers containing an azide moiety.

For example, mannuronate and guluronate residues (A) can amidated byreaction with an amine substituted with an azide moiety (for example,11-Azido-3,6,9-trioxaundecan-1-amine) in the presence of a carbodiimideand DMAP, forming azide-functionalized modified monomer F in a singlesynthetic step. Alternatively, mannuronate and guluronate residues (A)can amidated by reaction with an amine substituted with any moiety whichcan be readily transformed into an azide. For example, mannuronate andguluronate residues can be amidated by reaction with 4-iodobenzylaminein the presence of a carbodiimide and DMAP, forming iodo-functionalizedmonomer D. The iodine moiety can then be readily converted to the azide,for example by treatment with sodium azide.

Subsequently, the azide-functionalized monomers can be reacted with amolecule containing an alkyne functionality. For example,azide-functionalized monomers F and E can be reacted with a moleculecontaining a terminal alkyne functionality in the presence of acopper(I) catalyst (formed in situ by the reduction of CuSO₄ with sodiumascorbate), forming covalently modified monomers G and H.

Preferred alkynes for use as reagents in 1,3-dipolarcycloadditionreactions include those shown below.

3. Modification Via the Hydroxyl Moiety of the Mannuronate andGuluronate Monomers

Mannuronate and guluronate monomers contain hydroxyl moieties which canserve as a point of covalent modification. In preferred embodiments, thehydroxyl moieties of mannuronate and guluronate residues (1) are reactedas shown in Scheme 3.

Mannuronate and guluronate residues (A) can be phosphorylated by avariety of methods known in the art, forming covalently modified monomerI. For example, mannuronate and guluronate residues can bephosphorylated by reaction with I—PO(OR₅)₂ in the presence of pyridine(Stowell, J. K. and Widlanski, T. S. Tetrahedron Let. 1995; 36(11):1825-1826.).

Mannuronate and guluronate residues (A) can also be converted to acyclic acetal using procedures known in the art. For example, a cyclicacetal can be formed by reaction of mannuronate and guluronate residueswith any suitable ketone (R₂—CO—R₃) in acidic conditions.

4. Methods for Preparing Multiply Modified Alginate Polymers

In the case of singularly modified alginate polymers, only a singlereaction or sequence of reactions is performed, introducing one type ofcovalently modified monomer.

In the case of multiply modified alginate polymers, one or morereactions are performed to introduce multiple different types ofcovalently modified monomers into the modified alginate polymer. In someembodiments, multiply modified alginate polymers are prepared usingmultiple sequential synthetic reactions. For example, the multiplymodified alginate polymer shown below can be prepared using twosequential reactions: (1) amidation of mannuronate and guluronatemonomers with methylamine in the presence of CDMT and NMM; and (2)esterification of mannuronate and guluronate residues with ethanol inthe presence of CDMT and NMM.

In alternative embodiments, multiply modified alginate polymers can beprepared using a ‘one-pot’ synthesis. In these embodiments, multiplecovalently modified monomers are introduced into the alginate polymer ina single synthetic step. For example, the multiply modified alginatepolymer shown above can alternatively be prepared in a single syntheticstep by reacting an alginate polymer with methylamine and ethanol in thepresence of CDMT and NMM.

Any type or form of modified alginate, any type or form of alginatemodification, and any type or form of reagent for modifying alginate canbe, independently and in any combination, specifically included orexcluded in any of the disclosed modified alginates, alginatemodifications, reagents for alginate modifications, methods, and kits,and in any context, combination, or use. For example, any type or formof esterification reagent, amidation reagent, click reagent,alkyne-containing reagent, azide-containing reagent, phosphorylatingreagent, and ketone reagent, such as those described above and in theexamples, can be, independently and in any combination, specificallyincluded or excluded from use to modify alginates, and any alginatemodifications and any modified alginates that include or are based onsuch reagents can be, independently and in any combination, specificallyincluded or excluded in any of the disclosed modified alginates,alginate modifications, reagents for alginate modifications, methods,and kits, and in any context, combination, or use.

As another example, any of the reagents described in Table 2 can be,independently and in any combination, specifically included or excludedfrom use to modify alginates, and any alginate modifications and anymodified alginates that include or are based on the reagents describedin Table 2 can be, independently and in any combination, specificallyincluded or excluded in any of the disclosed modified alginates,alginate modifications, reagents for alginate modifications, methods,and kits, and in any context, combination, or use. For example, all ofthe reagents described in Table 2 in combination but excluding reagentY3 can be specifically included or excluded from use to modifyalginates, and any alginate modifications and any modified alginatesthat include or are based on all the reagents described in Table 2 incombination but excluding reagent Y3 can be specifically included orexcluded in any of the disclosed modified alginates, alginatemodifications, reagents for alginate modifications, methods, and kits,and in any context, combination, or use.

As another example, any modified alginate, alginate modification, orreagents for alginate modification described in U.S. Patent ApplicationNo. 20120308650 can be, independently and in any combination,specifically included or excluded. U.S. Patent Application PublicationNo. 20120308650 is hereby incorporated herein by reference in itsentirety, and specifically for its description of modified alginates,alginate modifications, and reagents for alginate modifications. Any ofthe R group substituents for any of the R groups described herein canbe, independently and in any combination, specifically included orexcluded as an option or as the choice for the respective R group.

D. Purification of Alginates

Commercial alginates are generally obtained from algae. Crude alginatesfrom seaweed contain numerous contaminants, including polyphenols,proteins, and endotoxins (de Vos, P, et al. Biomaterials 2006; 27:5603-5617). The presence of these impurities has been shown to limit thebiocompatibility of implanted alginates.

To optimize the biocompatibility of the chemically modified alginatesdescribed herein, a rigorous purification methodology was developed toeliminate potentially irritating impurities. In preferred embodiments,ultra-pure low viscosity alginate (UPVLVG, FMC Biopolymer) was used as asubstrate for covalent modification. Following each covalentmodification, the modified alginates were filtered through modifiedsilica columns, for example cyano-modified silica columns, aimed atcapturing bulk organic impurities. Finally, after covalent modificationof the alginate polymer is complete, the modified alginates are dialyzedto remove any remaining small-molecule or low molecular weightimpurities. In a preferred method, the modified alginates are dialyzedagainst 10,000 molecular weight cut-off (MWCO) membrane to remove anyremaining small-molecule impurities.

The purity of the modified alginates can be determined by ¹H NMRanalysis. In such an analysis, the ¹H NMR spectra of the modifiedalginate polymer is collected, and peaks corresponding to the modifiedalginate polymer and to any impurities are integrated to determine therelative quantity of each species in the sample. In some embodiments,the purity of the modified alginate polymer, as determined by ¹H NMR, isgreater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Inpreferred embodiments, the purity of the modified alginate polymer, asdetermined by ¹H NMR, is greater than 90%, more preferably greater than95%.

III. Biological Materials

Biological material for encapsulation in the disclosed alginates can beany biological substance. For example, the biological material can betissue, cells, biological micromolecules, or biological macromolecules.Examples of biological macromolecules include nucleotides, amino acids,cofactors, and hormones. Examples of biological macromolecules includenucleic acids, polypeptides, proteins, and polysaccharides. Examples ofproteins include enzymes, receptors, secretory proteins, structuralproteins, signaling proteins, hormones, and ligands. Any class, type,form, or particular biological material can be used together with anyother classes, types, forms, or particular biological materials.

A. Cells

The cell type chosen for encapsulation in the disclosed compositionsdepends on the desired therapeutic effect. The cells may be from thepatient (autologous cells), from another donor of the same species(allogeneic cells), or from another species (xenogeneic). Xenogeneiccells are easily accessible, but the potential for rejection and thedanger of possible transmission of viruses to the patient restrictstheir clinical application. Any of these types of cells can be fromnatural sources, stem cells, derived cells, or genetically engineeredcell.

In some embodiments, the cells secrete a therapeutically effectivesubstance, such as a protein or nucleic acid. In some embodiments, thecells produce a metabolic product. In some embodiments, the cellsmetabolize toxic substances. In some embodiments, the cells formstructural tissues, such as skin, bone, cartilage, blood vessels, ormuscle. In some embodiments, the cells are natural, such as islet cellsthat naturally secrete insulin, or hepatocytes that naturally detoxify.In some embodiments, the cells are genetically engineered to express aheterologous protein or nucleic acid and/or overexpress an endogenousprotein or nucleic acid.

Types of cells for encapsulation in the disclosed compositions includecells from natural sources, such as cells from xenotissue, cells from acadaver, and primary cells; stem cells, such as embryonic stem cells,mesenchymal stem cells, and induced pluripotent stem cells; derivedcells, such as cells derived from stem cells, cells from a cell line,reprogrammed cells, reprogrammed stem cells, and cells derived fromreprogrammed stem cells; and genetically engineered cells, such as cellsgenetically engineered to express a protein or nucleic acid, cellsgenetically engineered to produce a metabolic product, and cellsgenetically engineered to metabolize toxic substances.

Types of cells for encapsulation in the disclosed compositions includehepatocytes, islet cells, parathyroid cells, endocrine cells, cells ofintestinal origin, cells derived from the kidney, and other cells actingprimarily to synthesize and secret, or to metabolize materials. Apreferred cell type is a pancreatic islet cell or otherinsulin-producing cell. Hormone-producing cells can produce one or morehormones, such as insulin, parathyroid hormone, anti-diuretic hormone,oxytocin, growth hormone, prolactin, thyroid stimulating hormone,adrenocorticotropic hormone, follicle-stimulating hormone, lutenizinghormone, thyroxine, calcitonin, aldosterone, cortisol, epinephrine,glucagon, estrogen, progesterone, and testosterone. Geneticallyengineered cells are also suitable for encapsulation according to thedisclosed methods. In some embodiments, the cells are engineered toproduce one or more hormones, such as insulin, parathyroid hormone,anti-diuretic hormone, oxytocin, growth hormone, prolactin, thyroidstimulating hormone, adrenocorticotropic hormone, follicle-stimulatinghormone, lutenizing hormone, thyroxine, calcitonin, aldosterone,cortisol, epinephrine, glucagon, estrogen, progesterone, andtestosterone. In some embodiments, the cells are engineered to secreteblood clotting factors (e.g., for hemophilia treatment) or to secretegrowth hormones. In some embodiments, the cells are contained in naturalor bioengineered tissue. For example, the cells for encapsulation are insome embodiments a bioartificial renal glomerulus. In some embodiments,the cells are suitable for transplantation into the central nervoussystem for treatment of neurodegenerative disease.

Cells can be obtained directly from a donor, from cell culture of cellsfrom a donor, or from established cell culture lines. In the preferredembodiments, cells are obtained directly from a donor, washed andimplanted directly in combination with the polymeric material. The cellsare cultured using techniques known to those skilled in the art oftissue culture.

Cell viability can be assessed using standard techniques, such ashistology and fluorescent microscopy. The function of the implantedcells can be determined using a combination of these techniques andfunctional assays. For example, in the case of hepatocytes, in vivoliver function studies can be performed by placing a cannula into therecipient's common bile duct. Bile can then be collected in increments.Bile pigments can be analyzed by high pressure liquid chromatographylooking for underivatized tetrapyrroles or by thin layer chromatographyafter being converted to azodipyrroles by reaction with diazotizedazodipyrroles ethylanthranilate either with or without treatment withP-glucuronidase. Diconjugated and monoconjugated bilirubin can also bedetermined by thin layer chromatography after alkalinemethanolysis ofconjugated bile pigments. In general, as the number of functioningtransplanted hepatocytes increases, the levels of conjugated bilirubinwill increase. Simple liver function tests can also be done on bloodsamples, such as albumin production. Analogous organ function studiescan be conducted using techniques known to those skilled in the art, asrequired to determine the extent of cell function after implantation.For example, pancreatic islet cells and other insulin-producing cellscan be implanted to achieve glucose regulation by appropriate secretionof insulin. Other endocrine tissues and cells can also be implanted.

The site, or sites, where cells are to be implanted is determined basedon individual need, as is the requisite number of cells. For cellsreplacing or supplementing organ or gland function (for example,hepatocytes or islet cells), the mixture can be injected into themesentery, subcutaneous tissue, retroperitoneum, properitoneal space,and intramuscular space.

The amount and density of cells encapsulated in the disclosedcompositions, such as capsules and microcapsules, will vary depending onthe choice of cell, hydrogel, and site of implantation. In someembodiments, the single cells are present in the hydrogel at aconcentration of 0.1×10⁶ to 4×10⁶ cells/ml, preferred 0.5×10⁶ to 2×10⁶cells/mi. In other embodiments, the cells are present as cellaggregates. For example, islet cell aggregates (or whole islets)preferably contain about 1500-2000 cells for each aggregate of 150 μmdiameter, which is defined as one islet equivalent (IE). Therefore, insome embodiments, islet cells are present at a concentration of100-10000 IE/ml, preferably 200-3,000 IE/ml, more preferably 500-1500IE/ml.

1. Islet Cells and Other Insulin-Producing Cells

In preferred embodiments, the disclosed compositions contain islet cellsor other insulin-producing cells. Methods of isolating pancreatic isletcells are known in the art. Field et al., Transplantation 61:1554(1996); Linetsky et al., Diabetes 46:1120 (1997). Fresh pancreatictissue can be divided by mincing, teasing, comminution and/orcollagenase digestion. The islets can then be isolated fromcontaminating cells and materials by washing, filtering, centrifuging orpicking procedures. Methods and apparatus for isolating and purifyingislet cells are described in U.S. Pat. No. 5,447,863 to Langley, U.S.Pat. No. 5,322,790 to Scharp et al., U.S. Pat. No. 5,273,904 to Langley,and U.S. Pat. No. 4,868,121 to Scharp et al. The isolated pancreaticcells may optionally be cultured prior to microencapsulation, using anysuitable method of culturing islet cells as is known in the art. Seee.g., U.S. Pat. No. 5,821,121 to Brothers. Isolated cells may becultured in a medium under conditions that helps to eliminate antigeniccomponents. Insulin-producing cells can also be derived from stem cellsand cell lines and can be cells genetically engineered to produceinsulin.

2. Genetically Engineered Cells

In some embodiments, the disclosed compositions contain cellsgenetically engineered to produce a protein or nucleic acid (e.g., atherapeutic protein or nucleic acid). In these embodiments, the cell canbe, for example, a stem cell (e.g., pluripotent), a progenitor cell(e.g., multipotent or oligopotent), or a terminally differentiated cell(i.e., unipotent). Any of the disclosed cell types can be geneticallyengineered. The cell can be engineered, for example, to contain anucleic acid encoding, for example, a polynucleotide such miRNA or RNAior a polynucleotide encoding a protein. The nucleic acid can be, forexample, integrated into the cells genomic DNA for stable expression orcan be, for example, in an expression vector (e.g., plasmid DNA). Thepolynucleotide or protein can be selected based on the disease to betreated (or effect to be achieved) and the site of transplantation orimplantation. In some embodiments, the polynucleotide or protein isanti-neoplastic. In other embodiments, the polynucleotide or protein isa hormone, growth factor, or enzyme.

B. Hormones

Hormones to be included in the disclosed capsules or, most preferably,produced from cells encapsulated in the disclosed capsules can be anyhome of interest.

Examples of endocrine hormones include Anti-diuretic Hormone (ADH),which is produced by the posterior pituitary, targets the kidneys, andaffects water balance and blood pressure; Oxytocin, which is produced bythe posterior pituitary, targets the uterus, breasts, and stimulatesuterine contractions and milk secretion; Growth Hormone (GH), which isproduced by the anterior pituitary, targets the body cells, bones,muscles, and affects growth and development; Prolactin, which isproduced by the anterior pituitary, targets the breasts, and maintainsmilk secretions; Thyroid Stimulating Hormone (TSH), which is produced bythe anterior pituitary, targets the thyroid, and regulates thyroidhormones; Adrenocorticotropic Hormone (ACTH), which is produced by theanterior pituitary, targets the adrenal cortex, and regulates adrenalcortex hormones; Follicle-Stimulating Hormone (FSH), which is producedby the anterior pituitary, targets the ovaries/testes, and stimulatesegg and sperm production; Lutenizing Hormone (LH), which is produced bythe anterior pituitary, targets the ovaries/testes, and stimulatesovulation and sex hormone release; Thyroxine, which is produced by thethyroid, targets the body cells, and regulates metabolism; Calcitonin,which is produced by the thyroid, targets the adrenal cortex, and lowersblood calcium; Parathyroid Hormone, which is produced by theparathyroid, targets the bone matrix, and raises blood calcium;Aldosterone, which is produced by the adrenal cortex, targets thekidney, and regulates water balance; Cortisol, which is produced by theadrenal cortex, targets the body cells, and weakens immune system andstress responses; Epinephrine, which is produced by the adrenal medulla,targets the heart, lungs, liver, and body cells, and affects primary“fight or flight” responses; Glucagon, which is produced by thepancreas, targets the liver body, and raises blood glucose level;Insulin, which is produced by the pancreas, targets body cells, andlowers blood glucose level; Estrogen, which is produced by the ovaries,targets the reproductive system, and affects puberty, menstrual, anddevelopment of gonads; Progesterone, which is produced by the ovaries,targets the reproductive system, and affects puberty, menstrual cycle,and development of gonads; and Testosterone, which is produced by theadrenal gland, testes, targets the reproductive system, and affectspuberty, development of gonads, and sperm.

IV. Assays for the Characterization of Modified Alginate Polymers

The covalent modification of alginate polymers alters the physiochemicalproperties and biological compatibility of the alginate polymer.

In some embodiments, a hydrogel formation assay is used to quantify thestability of hydrogels formed from alginates or modified alginates. Inpreferred embodiments, the hydrogel formation assay is used as ascreening tool to identify modified alginates capable of forming stablehydrogels.

In vivo assays can be useful to characterize the biocompatibility ofmodified alginate polymers. In some embodiments, the high throughput invivo biocompatibility assay described herein is used to identifymodified alginates which induce a lower foreign body response thanunmodified alginate.

Further described herein is an in vivo method for quantifying thebiocompatibility of modified alginates.

The assays can be used to assess the suitability and biocompatibility ofboth modified and unmodified alginates for certain applications.

A. High Throughput Hydrogel Formation Assay

Covalent modification of the alginates affects the physical propertiesof the alginate, including the ability of the alginate to form hydrogelssuitable for the encapsulation of cells and biomolecules.

The gel-forming assay exploits the ability of hydrogels to trapfluorescent compounds, and differentially retain the fluorophores uponwashing based on the stability of the hydrogel. In this assay, ahydrogel formed by ionically crosslinking a candidate modified alginatein aqueous solution containing a dissolved fluorophore. A variety offluorophores can be used in this assay. In preferred embodiments, thefluorophores possess emission maxima between 480 and 750 nm. Inpreferred embodiments, the fluorophore is a rhodamine dye possessing anemission maximum between 550 and 600 nm.

After crosslinking, the hydrogel is repeatedly washed with water.Candidate modified alginates which do not efficiently crosslink arewashed away along with any fluorophore present. Modified alginates whichefficiently crosslink retain the fluorophore during washes. Accordingly,by measuring the fluorescence of modified alginate hydrogels afterwashing, modified alginates capable of forming stable hydrogels can bereadily identified.

In some embodiments, the relative fluorescence intensity values measuredfor a modified alginate are compared relative to fluorescence levelsmeasured for the negative control and unmodified alginate to determineif the modified alginate is capable of forming hydrogels. In alternativeembodiments, the hydrogel formation assay described herein is used toquantify the stability of hydrogels formed from alginates or modifiedalginates. In these embodiments, the fluorescence intensity measured fora modified alginate is used to indicate the stability of the hydrogelformed by the alginate.

In preferred embodiments, the modified alginate polymer forms hydrogelssuch that the fluorescence intensity measured using the high throughputhydrogel formation assay described herein is greater than 10,000,15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or55,000. In preferred embodiments, the modified alginate polymer formshydrogels such that the fluorescence intensity measured using the highthroughput hydrogel formation assay described herein is greater than15,000. In preferred embodiments, the modified alginate polymer formshydrogels such that the fluorescence intensity measured using the highthroughput hydrogel formation assay described herein is between 15,000and 55,000, more preferably between 25,000 and 55,000.

B. High Throughput In Vivo Biocompatibility Assay

Current biocompatibility analysis methods are slow and requirehistological analysis. Described herein is a high throughput in vivobiocompatibility assay, useful for assessing the relativebiocompatibility of alginate polymers.

In the high throughput in vivo biocompatibility assay described herein,modified alginate polymers and an unmodified alginate control areinjected in an array format on the back of an animal test subject tofacilitate high-throughput screening. In preferred embodiments, theanimal test subject is a mouse. After injection, cathepsin activity atthe point of injection of the modified alginates was compared tocathepsin activity at the point of injection the unmodified alginate tocompare the foreign body response to the implanted alginates using invivo fluorescence imaging. In preferred embodiments, thebiocompatibility of the materials was assessed at 14 days post injectionusing in vivo fluorescence imaging.

In preferred embodiments, the high throughput in vivo biocompatibilityassay described herein is used to identify modified alginates whichinduce a lower foreign body response than unmodified alginate. Thefluorescence intensity measured at the implantation site of modifiedalginates was compared with the fluorescence intensity measured at theimplantation site of an unmodified alginate. In preferred embodiments,modified alginates exhibiting smaller fluorescence intensity at theimplantation site than the fluorescence intensity measured at theimplantation site of unmodified alginates were qualitativelycharacterized as biocompatible. Conversely, modified alginatesexhibiting greater fluorescence intensity at the implantation site thanthe fluorescence intensity measured at the implantation site ofunmodified alginates were characterized as not biocompatible.

The high throughput in vivo biocompatibility assay described above canalso be used to characterize the ability of modified alginates to formmechanically stable hydrogels in vivo. In preferred embodiments, the invivo stability of the alginate gels was assessed at 28 days postinjection.

In preferred embodiments, modified alginates gels which remained at thesite of injection after 28 days were characterized as capable of formingmechanically stable hydrogels in vivo. Conversely, modified alginategels which were not present at the site of injection after 28 days weredeemed to not capable of forming mechanically stable hydrogels in vivo.

C. In Vivo Screening of Modified Alginates to Quantify biocompatibility

Further described herein is an in vivo method for quantifying thebiocompatibility of modified alginates.

In this method, a modified alginate polymers is injected on the back ofan animal test subject. In preferred embodiments, the animal testsubject is a mouse. After injection, cathepsin activity at the point ofinjection of the modified alginates was measured using in vivofluorescence assay. In preferred embodiments, the fluorescence assay wasconducted at 7 days post injection using in vivo fluorescence imaging.In preferred embodiments, the fluorescence intensity was measured andnormalized to the fluorescence response measured using unmodifiedalginate in order to quantify the biocompatibility of the modifiedalginates.

In preferred embodiments, the modified alginate polymer induces a lowerforeign body response than unmodified alginate (i.e. the fluorescenceresponse normalized to unmodified alginate is less that 100%). In someembodiments, the modified alginate polymer is biocompatible such thatthe fluorescence response normalized to unmodified alginate measuredusing the in vivo biocompatibility assay described herein is less than95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%. Inpreferred embodiments, the modified alginate polymer is biocompatiblesuch that the fluorescence response normalized to unmodified alginatemeasured using the in vivo biocompatibility assay described herein isless than 75%, more preferably less than 65%, and most preferably lessthan 50%.

V. Methods of Use

Alginates are used in a variety of applications in the food,pharmaceutical, cosmetic, agriculture, printing, and textile industries.Alginates are widely employed in the food industry as thickening,gelling, stabilizing, bodying, suspending, and emulsifying agents.Alginates can be used as a matrix to control the delivery oftherapeutic, prophylactic, and/or diagnostic agents. Alginates can beincorporated in pharmaceutical compositions as excipients, where theycan act as viscosifiers, suspension agents, emulsifiers, binders, anddisintigrants. Alginate also used as a dental impression material,component of wound dressings, and as a printing agent. One of ordinaryskill in the art will recognize that the modified alginates disclosedherein can be used in any application for which alginates are currentlyemployed.

It is specifically contemplated that modified alginates described hereincan be used in applications where improved biocompatibility and physicalproperties (such as being anti-fibrotic), as compared to commerciallyavailable alginates, are preferred.

A. Encapsulation of Cells

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. See, for example, inU.S. Pat. No. 4,352,883 to Lim. In the Lim process, an aqueous solutioncontaining the biological materials to be encapsulated is suspended in asolution of a water soluble polymer, the suspension is formed intodroplets which are configured into discrete capsules by contact withmultivalent cations, then the surface of the capsules is crosslinkedwith polyamino acids to form a semipermeable membrane around theencapsulated materials.

The water soluble polymer with charged side groups is crosslinked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups or multivalent anions if the polymer has basicside groups. The preferred cations for cross-linking of the polymerswith acidic side groups to form a hydrogel are divalent and trivalentcations such as copper, calcium, aluminum, magnesium, strontium, barium,and tin, although di-, tri- or tetra-functional organic cations such asalkylammonium salts, e.g., R₃N+--\/\/\/--+NR₃ can also be used. Aqueoussolutions of the salts of these cations are added to the polymers toform soft, highly swollen hydrogels and membranes. The higher theconcentration of cation or the higher the valence, the greater thedegree of cross-linking of the polymer. Concentrations from as low as0.005 M have been demonstrated to cross-link the polymer. Higherconcentrations are limited by the solubility of the salt.

The preferred anions for cross-linking of polymers containing basicsidechains to form a hydrogel are divalent and trivalent anions such aslow molecular weight dicarboxylic acids, for example, terepthalic acid,sulfate ions and carbonate ions. Aqueous solutions of the salts of theseanions are added to the polymers to form soft, highly swollen hydrogelsand membranes, as described with respect to cations.

A variety of polycations can be used to complex and thereby stabilizethe polymer hydrogel into a semi-permeable surface membrane. Examples ofmaterials that can be used include polymers having basic reactive groupssuch as amine or imine groups, having a preferred molecular weightbetween 3,000 and 100,000, such as polyethylenimine and polylysine.These are commercially available. One polycation is poly(L-lysine);examples of synthetic polyamines are: polyethyleneimine,poly(vinylamine), and poly(allyl amine). There are also naturalpolycations such as the polysaccharide, chitosan.

Polyanions that can be used to form a semi-permeable membrane byreaction with basic surface groups on the polymer hydrogel includepolymers and copolymers of acrylic acid, methacrylic acid, and otherderivatives of acrylic acid, polymers with pendant SO₃H groups such assulfonated polystyrene, and polystyrene with carboxylic acid groups.

In a preferred method, cells are encapsulated in a modified alginatepolymer. In a preferred embodiment, modified alginate capsules arefabricated from solution of modified alginate containing suspended cellsusing the encapsulator (such as an Inotech encapsulator). In someembodiments, modified alginates are ionically crosslinked with apolyvalent cation, such as Ca²⁺, Ba²⁺, or Sr²⁺. In particularlypreferred embodiments, the modified alginate is crosslinked using BaCl₂.In some embodiments, the capsules are further purified after formation.In preferred embodiments, the capsules are washed with, for example,HEPES solution, Krebs solution, and/or RPMI-1640 medium.

Cells can be obtained directed from a donor, from cell culture of cellsfrom a donor, or from established cell culture lines. In the preferredembodiments, cells are obtained directly from a donor, washed andimplanted directly in combination with the polymeric material. The cellsare cultured using techniques known to those skilled in the art oftissue culture. In the preferred embodiment, the cells areautologous—i.e., derived from the individual into which the cells are tobe transplanted, but may be allogeneic or heterologous.

Cell attachment and viability can be assessed using scanning electronmicroscopy, histology, and quantitative assessment with radioisotopes.The function of the implanted cells can be determined using acombination of the above-techniques and functional assays. For example,in the case of hepatocytes, in vivo liver function studies can beperformed by placing a cannula into the recipient's common bile duct.Bile can then be collected in increments. Bile pigments can be analyzedby high pressure liquid chromatography looking for underivatizedtetrapyrroles or by thin layer chromatography after being converted toazodipyrroles by reaction with diazotized azodipyrrolesethylanthranilate either with or without treatment with P-glucuronidase.Diconjugated and monoconjugated bilirubin can also be determined by thinlayer chromatography after alkalinemethanolysis of conjugated bilepigments. In general, as the number of functioning transplantedhepatocytes increases, the levels of conjugated bilirubin will increase.Simple liver function tests can also be done on blood samples, such asalbumin production. Analogous organ function studies can be conductedusing techniques known to those skilled in the art, as required todetermine the extent of cell function after implantation. For example,islet cells of the pancreas may be delivered in a similar fashion tothat specifically used to implant hepatocytes, to achieve glucoseregulation by appropriate secretion of insulin to cure diabetes. Otherendocrine tissues can also be implanted. Studies using labeled glucoseas well as studies using protein assays can be performed to quantitatecell mass on the polymer scaffolds. These studies of cell mass can thenbe correlated with cell functional studies to determine what theappropriate cell mass is. In the case of chondrocytes, function isdefined as providing appropriate structural support for the surroundingattached tissues.

This technique can be used to provide multiple cell types, includinggenetically altered cells, within a three-dimensional scaffolding forthe efficient transfer of large number of cells and the promotion oftransplant engraftment for the purpose of creating a new tissue ortissue equivalent. It can also be used for immunoprotection of celltransplants while a new tissue or tissue equivalent is growing byexcluding the host immune system.

Examples of cells which can be implanted as described herein includechondrocytes and other cells that form cartilage, osteoblasts and othercells that form bone, muscle cells, fibroblasts, and organ cells. Asused herein, “organ cells” includes hepatocytes, islet cells, cells ofintestinal origin, cells derived from the kidney, and other cells actingprimarily to synthesize and secret, or to metabolize materials. Apreferred cell type is a pancreatic islet cell.

The polymeric matrix can be combined with humoral factors to promotecell transplantation and engraftment. For example, the polymeric matrixcan be combined with angiogenic factors, antibiotics,antiinflammatories, growth factors, compounds which inducedifferentiation, and other factors which are known to those skilled inthe art of cell culture.

For example, humoral factors could be mixed in a slow-release form withthe cell-alginate suspension prior to formation of implant fortransplantation. Alternatively, the hydrogel could be modified to bindhumoral factors or signal recognition sequences prior to combinationwith isolated cell suspension.

The techniques described herein can be used for delivery of manydifferent cell types to achieve different tissue structures. In thepreferred embodiment, the cells are mixed with the hydrogel solution andinjected directly into a site where it is desired to implant the cells,prior to hardening of the hydrogel. However, the matrix may also bemolded and implanted in one or more different areas of the body to suita particular application. This application is particularly relevantwhere a specific structural design is desired or where the area intowhich the cells are to be implanted lacks specific structure or supportto facilitate growth and proliferation of the cells.

The site, or sites, where cells are to be implanted is determined basedon individual need, as is the requisite number of cells. For cellshaving organ function, for example, hepatocytes or islet cells, themixture can be injected into the mesentery, subcutaneous tissue,retroperitoneum, properitoneal space, and intramuscular space. Forformation of cartilage, the cells are injected into the site wherecartilage formation is desired. One could also apply an external mold toshape the injected solution. Additionally, by controlling the rate ofpolymerization, it is possible to mold the cell-hydrogel injectedimplant like one would mold clay. Alternatively, the mixture can beinjected into a mold, the hydrogel allowed to harden, then the materialimplanted.

B. Coating Products and Surfaces

Medical products can be coated with the disclosed modified alginatepolymers using a variety of techniques, examples of which includespraying, dipping, and brush coating. Polymer coatings are typicallyapplied to the surface to be coated by dissolving a polymer in anappropriate, preferably organic solvent, and applying by spraying,brushing, dipping, painting, or other similar technique. The coatingsare deposited on the surface and associate with the surfaces vianon-covalent interactions. The coated products and surfaces that resultare specifically contemplated and disclosed.

In some embodiments, the surface may be pretreated with an appropriatesolution or suspension to modify the properties of the surface, andthereby strengthen the non-covalent interactions between the modifiedsurface and the coating.

The polymer solution is applied to a surface at an appropriatetemperature and for a sufficient period of time to form a coating on thesurface, wherein the coating is effective in forming an anti-fibroticsurface. Typical temperatures include room temperature, although highertemperatures may be used. Typical time periods include 5 minutes orless, 30 minutes or less, 60 minutes or less, and 120 minutes or less.In some embodiments the solution can be applied for 120 minutes orlonger to form a coating with the desired anti-fibrotic activity.However, preferably shorter time periods are used. Anti-fibroticactivity can be measured in any of the ways disclosed herein or known inthe art. Preferably the anti-fibrotic activity can be the foreign bodyresponse determined as described herein.

The disclosed modified alginate polymers can be covalently ornon-covalently associated with the products, devices, and surfaces. Forthose embodiments where the modified alginate polymer is covalentlyassociated with the product, device, or surface, the polymer can beattached to the product, device, or surface by, for example,functionalizing the product, device, or surface with a reactionfunctional group, such as a nucleophilic group, and reacting thenucleophilic group with a reaction functional group on the polymer, suchas an electrophilic group. Alternatively, the polymer can befunctionalized with a nucleophilic group which is reacted with anelectrophilic group on the product, device, or surface.

In particular embodiments, the modified alginate polymer isnon-covalently associated with the product, device, or surface. Thepolymer can be applied to the product, device, or surface by spraying,wetting, immersing, dipping, painting, bonding or adhering or otherwiseproviding a product, device, or surface with a compound with themodified alginate polymer. In one embodiment, the polymer is applied byspraying, painting, or dipping or immersing. For example, a polymerpaint can be prepared by dissolving the modified alginate polymer in asuitable solvent (generally aqueous), and optionally sonicating thesolution to ensure the polymer is completely dissolved. The product,device, or surface to be coated can be immersed in the polymer solutionfor a suitable period of time, e.g., 5 seconds, followed by drying, suchas air drying. The procedure can be repeated as many times as necessaryto achieve adequate coverage. The thickness of the coating is generallyfrom about 1 nm to about 1 cm, preferably from about 10 nm to 1 mm, morepreferably from about 100 nm to about 100 microns.

The coating can be applied at the time the product, device, or surfaceis manufactured or can be applied subsequent to manufacture of theproduct, device, or surface. In some embodiments, the coating is appliedto the product, device, or surface immediately prior to use of theproduct, device, or surface. This is referred to an intraoperativecoating. “Immediately prior”, as used herein, mean within 1, 2, 5, 10,15, 20, 30, 45, 60, 75, 90, 120, 150, 180 minutes or greater ofimplantation or use. In some embodiments, the product, device, orsurface is coated at the hospital, e.g., in the operating room, with 20,15, 10, or 5 minutes of implantation or use. Coating immediately priorto use may overcome limitations of products, devices, and surfacescoated at the time of manufacture, such as damage of the coating duringstorage and/or transportation of the product, device, or surface and/ordecrease in the efficacy of the coating over time as the coating isexposed to environmental conditions, which may be harsh (e.g., hightemps, humidity, exposure to UV light, etc.).

The coated medical products can be used for the known uses and purposesof uncoated or differently coated forms of the medical products.

1. Medical Products

Medical products useful for coating include any types of medical devicesused, at least in part, for implantation in the body of a patient.Examples include, but are not limited to, implants, implantable medicalproducts, implantable devices, catheters and other tubes (includingurological and biliary tubes, endotracheal tubes, wound drain tubes,needle injection catheters, peripherably insertable central venouscatheters, dialysis catheters, long term tunneled central venouscatheters peripheral venous catheters, short term central venouscatheters, arterial catheters, pulmonary catheters, Swan-Ganz catheters,urinary catheters, peritoneal catheters), vascular catheter ports, bloodclot filters, urinary devices (including long term urinary devices,tissue bonding urinary devices, artificial urinary sphincters, urinarydilators), shunts (including ventricular or arterio-venous shunts, stenttransplants, biliary stents, intestinal stents, bronchial stents,esophageal stents, ureteral stents, and hydrocephalus shunts), balloons,pacemakers, implantable defibrillators, orthopedic products (includingpins, plates, screws, and implants), transplants (including organs,vascular transplants, vessels, aortas, heart valves, and organreplacement parts), prostheses (including breast implants, penileprostheses, vascular grafting prostheses, heart valves, artificialjoints, artificial larynxes, otological implants, artificial hearts,artificial blood vessels, and artificial kidneys), aneurysm-fillingcoils and other coil devices, transmyocardial revascularization devices,percutaneous myocardial revascularization devices, tubes, fibers, hollowfibers, membranes, blood containers, titer plates, adsorber media,dialyzers, connecting pieces, sensors, valves, endoscopes, filters, pumpchambers, scalpels, needles, scissors (and other devices used ininvasive surgical, therapeutic, or diagnostic procedures), and othermedical products and devices intended to have anti-fibrotic properties.The expression “medical products” is broad and refers in particular toproducts that come in contact with blood briefly (e.g., endoscopes) orpermanently (e.g., stents).

Useful medical products are balloon catheters and endovascularprostheses, in particular stents. Stents of a conventional design have afiligree support structure composed of metallic struts. The supportstructure is initially provided in an unexpanded state for insertioninto the body, and is then widened into an expanded state at theapplication site. The stent can be coated before or after it is crimpedonto a balloon. A wide variety of medical endoprostheses or medicalproducts or implants for highly diverse applications and are known. Theyare used, for example, to support vessels, hollow organs, and ductalsystems (endovascular implants), to attach and temporarily affix tissueimplants and tissue transplants, and for orthopedic purposes such aspins, plates, or screws.

The modified alginate polymers can be applied to, absorbed into, orcoupled to, a variety of different substrates and surfaces. Examples ofsuitable materials include metals, metallic materials, ceramics,polymers, fibers, inert materials such as silicon, and combinationsthereof.

Suitable polymeric materials include, but are not limited to, styreneand substituted styrenes, ethylene, propylene, poly(urethane)s,acrylates and methacrylates, acrylamides and methacrylamides,polyesters, polysiloxanes, polyethers, poly(orthoester),poly(carbonates), poly(hydroxyalkanoate)s, copolymers thereof, andcombinations thereof.

Substrates can be in the form of, or form part of, films, particles(nanoparticles, microparticles, or millimeter diameter beads), fibers(wound dressings, bandages, gauze, tape, pads, sponges, including wovenand non-woven sponges and those designed specifically for dental orophthalmic surgeries), sensors, pacemaker leads, catheters, stents,contact lenses, bone implants (hip replacements, pins, rivets, plates,bone cement, etc.), or tissue regeneration or cell culture devices, orother medical devices used within or in contact with the body.

Implants coated with modified alginate polymer coatings are describedherein. “Implants” are any object intended for placement in the body ofa mammal, such as a human, that is not a living tissue. Implants are aform of medical product. Implants include naturally derived objects thathave been processed so that their living tissues have been devitalized.As an example, bone grafts can be processed so that their living cellsare removed, but so that their shape is retained to serve as a templatefor ingrowth of bone from a host. As another example, naturallyoccurring coral can be processed to yield hydroxyapatite preparationsthat can be applied to the body for certain orthopedic and dentaltherapies. An implant can also be an article comprising artificialcomponents. The term “implant” can be applied to the entire spectrum ofmedical devices intended for placement in a human body or that of amammal, including orthopedic applications, dental applications, ear,nose, and throat (“ENT”) applications, and cardiovascular applications.

In some embodiments, “implant” as used herein refers to a macroscopiccomposition including a device for implantation or a surface of a devicefor implantation and a modified alginate polymer coating. In theseembodiments, the term “implant” does not encompass nanoparticles and/ormicroparticles. “Macroscopic” as used herein generally refers todevices, implants, or compositions that can be viewed by the unaidedeye.

Examples of implantable medical devices and medical devices andmechanical structures that can use a bio-compatible coating include, butare not limited to, stents, conduits, scaffolds, cardiac valve rings,cardiovascular valves, pacemakers, hip replacement devices, implantedsensor devices, esophageal stents, heart implants, bio-compatiblelinings for heart valves, dialysis equipment and oxygenator tubing forheart-lung by-pass systems.

In general, a stent is a device, typically tubular in shape, that isinserted into a lumen of the body, such as a blood vessel or duct, toprevent or counteract a localized flow constriction. The purpose of astent, in some cases, is to mechanically prop open a bodily fluidconduit. Stents are often used to alleviate diminished blood flow toorgans and extremities in order to maintain adequate delivery ofoxygenated blood. The most common use of stents is in coronary arteries,but they are also widely used in other bodily conduits, such as, forexample, central and peripheral arteries and veins, bile ducts, theesophagus, colon, trachea, large bronchi, ureters, and urethra.Frequently, stents inserted into a lumen are capable of being expandedafter insertion or are self-expanding. For example, metal stents aredeployed into an occluded artery using a balloon catheter and expandedto restore blood flow. For example, stainless steel wire mesh stents arecommercially available from Boston Scientific, Natick, Mass.

In some embodiments, the implant is an orthopedic implant. An“orthopedic implant” is defined as an implant which replaces bone orprovides fixation to bone, replaces articulating surfaces of a joint,provides abutment for a prosthetic, or combinations thereof or assistsin replacing bone or providing fixation to bone, replacing articulatingsurfaces of a joint, providing abutment for a prosthetic, andcombinations thereof.

Orthopedic implants can be used to replace bone or provide fixation tobone, replace articulating surfaces of a joint, provide abutment for aprosthetic, or combinations thereof or assist in replacing bone orproviding fixation to bone, replacing articulating surfaces of a joint,providing abutment for a prosthetic, including dental applications, andcombinations thereof.

Suitable orthopedic implants include, but are not limited to, wire,Kirschner wire, bone plates, screws, pins, tacs, rods, nails, nuts,bolts, washers, spikes, buttons, wires, fracture plates, reconstructionand stabilizer devices, endo- and exoprostheses (articulating andnon-articulating), intraosseous transcutaneous prostheses, spacers,mesh, implant abutments, anchors, barbs, clamps, suture, interbodyfusion devices, tubes of any geometry, scaffolds, and combinationsthereof.

In other embodiments, the implant is an ear, nose, and/or throat (“ENT”)implant. Exemplary ENT implants include, but are not limited to, eartubes, endotracheal tubes, ventilation tubes, cochlear implants and boneanchored hearing devices.

In other embodiments, the implant is a cardiovascular implant. Exemplarycardiovascular implants are cardiac valves or alloplastic vessel wallsupports, total artificial heart implants, ventricular assist devices,vascular grafts, stents, electrical signal carrying devices such aspacemaker and neurological leads, defibrillator leads, and the like.

Implants can be prepared from a variety of materials. In someembodiments, the material is biocompatible. In some embodiments, thematerial is biocompatible and non-biodegradable. Exemplary materialsinclude metallic materials, metal oxides, polymeric materials, includingdegradable and non-degradable polymeric materials, ceramics, porcelains,glass, allogeneic, xenogenic bone or bone matrix; genetically engineeredbone; and combinations thereof.

Suitable metallic materials include, but are not limited to, metals andalloys based on titanium (such as nitinol, nickel titanium alloys,thermo-memory alloy materials), stainless steel, tantalum, palladium,zirconium, niobium, molybdenum, nickel-chrome, or certain cobalt alloysincluding cobalt-chromium and cobalt-chromium-nickel alloys such asELGILOY® and PHYNOX®. Useful examples include stainless steel grade 316(SS 316L) (comprised of Fe, <0.3% C, 16-18.5% Cr, 10-14% Ni, 2-3% Mo,<2% Mn, <1% Si, <0.45% P, and <0.03% S), tantalum, chromium molybdenumalloys, nickel-titanium alloys (such as nitinol) and cobalt chromiumalloys (such as MP35N, ASTM Material Designation: 35Co-35Ni-20Cr-10Mo).Typical metals currently in use for stents include SS 316L steel andMP35N. See also, “Comparing and Optimizing Co—Cr Tubing for StentApplications,” Poncin, P, Millet, C., Chevy, J, and Profit, J. L.,Materials & Processes for Medical Devices Conference, August 2004, ASMInternational.

Suitable ceramic materials include, but are not limited to, oxides,carbides, or nitrides of the transition elements such as titaniumoxides, hafnium oxides, iridium oxides, chromium oxides, aluminumoxides, and zirconium oxides. Silicon based materials, such as silica,may also be used.

Suitable polymeric materials include, but are not limited to,polystyrene and substituted polystyrenes, polyethylene, polypropylene,polyacetylene, polystyrene, TEFLON®, poly(vinyl chloride) (PVC),polyolefin copolymers, poly(urethane)s, polyacrylates andpolymethacrylates, polyacrylamides and polymethacrylamides, polyesters,polysiloxanes, polyethers, poly(orthoester), poly(carbonates),poly(hydroxyalkanoate)s, polyfluorocarbons, PEEK®, Teflon®(polytetrafluoroethylene, PTFE), silicones, epoxy resins, Kevlar®,Dacron® (a condensation polymer obtained from ethylene glycol andterephthalic acid), nylon, polyalkenes, phenolic resins, natural andsynthetic elastomers, adhesives and sealants, polyolefins, polysulfones,polyacrylonitrile, biopolymers such as polysaccharides and naturallatex, collagen, cellulosic polymers (e.g., alkyl celluloses, etc.),polysaccharides, poly(glycolic acid), poly(L-lactic acid) (PLLA), apolydioxanone (PDA), or racemic poly(lactic acid), polycarbonates,(e.g., polyamides (nylon); fluoroplastics, carbon fiber, and blends orcopolymers thereof.

The polymer can be covalently or non-covalently associated with thesurface; however, in particular embodiments, the polymer isnon-covalently associated with the surface. The polymer can be appliedby a variety of techniques in the art including, but not limited to,spraying, wetting, immersing, dipping, such as dip coating (e.g.,intraoperative dip coating), painting, or otherwise applying ahydrophobic, polycationic polymer to a surface of the implant.

A surface of a product adapted for use in a medical environment can becapable of sterilization using autoclaving, biocide exposure,irradiation, or gassing techniques, like ethylene oxide exposure.Surfaces found in medical environments include the inner and outeraspects of various instruments and devices, whether disposable orintended for repeated uses.

2. Hydrogels

Medical products can also be made of or using hydrogels. The disclosedmodified alginate polymers can form hydrogels for this and otherpurposes. Products made of other hydrogels can also be coated with thedisclosed modified alginate polymers. Thus, the disclosed modifiedalginate polymers can be used as a coating on a product or surface orcan be used as the product itself. Hydrogels are three-dimensional,hydrophilic, polymeric networks capable of imbibing large amounts ofwater or biological fluids (Peppas et al. Eur. J. Pharm. Biopharm. 2000,50, 27-46). These networks are composed of homopolymers or copolymers,and are insoluble due to the presence of chemical crosslinks or physicalcrosslinks, such as entanglements or crystallites. Hydrogels can beclassified as neutral or ionic, based in the nature of the side groups.In addition, they can be amorphous, semicrystalline, hydrogen-bondedstructures, supermolecular structures and hydrocolloidal aggregates(Peppas, N. A. Hydrogels. In: Biomaterials science: an introduction tomaterials in medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J.,Lemons, J. E., Eds; Academic Press, 1996, pp. 60-64; Peppas et al., Eur.J. Pharm. Biopharm. 2000, 50, 27-46). Hydrogels can be prepared fromsynthetic or natural monomers or polymers. Preferred hydrogels hereinare the disclosed modified alginate polymers.

Hydrogels can be prepared from synthetic polymers such as poly(acrylicacid) and its derivatives [e.g. poly(hydroxyethyl methacrylate)(pHEMA)], poly(N-isopropylacrylamide), poly(ethylene glycol) (PEG) andits copolymers and poly(vinyl alcohol) (PVA), among others (Bell andPeppas, Adv. Polym. Sci. 122:125-175 (1995); Peppas et al., Eur. J.Pharm. Biopharm. 50:27-46 (200); Lee and Mooney, Chem. Rev.101:1869-1879 (2001)). Hydrogels prepared from synthetic polymers are ingeneral non-degradable in physiologic conditions.

Hydrogels can also be prepared from natural polymers including, but notlimited to, polysaccharides, proteins, and peptides. The disclosedmodified alginate polymers are a preferred example. These networks arein general degraded in physiological conditions by chemical or enzymaticmeans.

In some embodiments, the hydrogel is non-degradable under relevant invitro and in vivo conditions. Stable hydrogel coatings are necessary forcertain applications including central venous catheters coating, heartvalves, pacemakers and stents coatings. In other cases, hydrogeldegradation may be a preferential approach such as in tissue engineeringconstructs.

In some embodiments, the hydrogel can be formed by dextran. Dextran is abacterial polysaccharide, consisting essentially of α-1,6 linkedD-glucopyranose residues with a few percent of α-1,2, α-1,3, orα-1,4-linked side chains. Dextran is widely used for biomedicalapplications due to its biocompatibility, low toxicity, relatively lowcost, and simple modification. This polysaccharide has been usedclinically for more than five decades as a plasma volume expander,peripheral flow promoter and antithrombolytic agent (Mehvar, R. J.Control. Release 2000, 69, 1-25). Furthermore, it has been used asmacromolecular carrier for delivery of drugs and proteins, primarily toincrease the longevity of therapeutic agents in the circulation. Dextrancan be modified with vinyl groups either by using chemical or enzymaticmeans to prepare gels (Ferreira et al. Biomaterials 2002, 23,3957-3967).

Dextran-based hydrogels prevent the adhesion of vascular endothelial,smooth muscle cells, and fibroblasts (Massia, S. P.; Stark, J. J.Biomed. Mater. Res. 2001, 56, 390-399. Ferreira et al. 2004, J. Biomed.Mater. Res. 68A, 584-596) and dextran surfaces prevent proteinadsorption (Österberg et al. J. Biomed. Mat. Res. 1995, 29, 741-747).

As described herein, the disclosed modified alginate polymers can beused to encapsulate cells. In some embodiments, the encapsulated cellscan be fabricated into a macrodevice. For example, in some embodiments,cells encapsulated in modified alginate hydrogel can be coated onto asurface, such as a planar surface. In some embodiments, capsulescontaining cells can be adhered to tissue of a subject using abiocompatible adhesive. In other embodiments, capsules containing cellscan be coated onto a medical device suitable for implantation.

C. Treatment of Diseases or Disorders

Encapsulated cells can be transplanted into a patient in need thereof totreat a disease or disorder. In some embodiments, the encapsulated cellsare obtained from a genetically non-identical member of the samespecies. In alternative embodiments, the encapsulated cells are obtainedfrom a different species than the patient. In preferred embodiments,hormone- or protein-secreting cells are encapsulated and transplantedinto a patient to treat a disease or disorder.

In preferred embodiments, the disease or disorder is caused by orinvolves the malfunction hormone- or protein-secreting cells in apatient. In a preferred embodiment, the disease or disorder is diabetes.

Medical products, devices, and surfaces coated with a modified alginatepolymer can be transplanted or implanted into a patient in need thereofto treat a disease or disorder.

The disclosed capsules, products, devices, and surfaces can remainsubstantially free of fibrotic effects, or can continue to exhibit areduced foreign body response, for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8, months, 9 months, 10 months, 11months, 1 year, 2 years, or longer after administration or implantation.

The disclosed capsules, products, devices, and surfaces can beadministered or implanted alone or in combination with any suitable drugor other therapy. Such drugs and therapies can also be separatelyadministered (i.e., used in parallel) during the time the capsules,products, devices, and surfaces are present in a patient. Although thedisclosed capsules, products, devices, and surfaces reduce fibrosis andimmune reaction to the capsules, products, devices, and surfaces, use ofanti-inflammatory and immune system suppressing drugs together with orin parallel with the capsules, products, devices, and surfaces is notexcluded. In preferred embodiments, however, the disclosed capsules,products, devices, and surfaces are used without the use ofanti-inflammatory and immune system suppressing drugs. In preferredembodiments, fibrosis remains reduced after the use, concentration,effect, or a combination thereof, of any anti-inflammatory or immunesystem suppressing drug that is used falls below an effective level. Forexample, fibrosis can remain reduced for 2 weeks, 3 weeks, 4 weeks, 5weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 2 months, 3 months,4 months, 5 months, 6 months, 7 months, 8, months, 9 months, 10 months,11 months, 1 year, 2 years, or longer after the use, concentration,effect, or a combination thereof, of any anti-inflammatory or immunesystem suppressing drug that is used falls below an effective level.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1: Combinatorial Synthesis of Chemically ModifiedAlginates

The determinate parameters governing material biocompatibility arepoorly understood. Accordingly, the rational design and synthesis ofmodified alginates possessing improved biocompatibility is challenging.In an effort to identify modified alginates with improvedbiocompatibility and physical properties, a combinatorial approach wasused to prepare a library of modified alginates possessing a range ofcovalent modifications.

1. General Combinatorial Strategy

A pool of twelve alcohols, nine amines, two amines used to introduce anazide moiety (one amine containing an azide moiety and one aminecontaining an iodide moiety to be converted to an azide moietysubsequent to amidation), and nineteen alkynes with a range of differentchemical structures, hydrophobicities/hydrophilicities, hydrogen-bondingpotentials, and charge states were selected as reagents for thecombinatorial synthesis of modified alginates. With the knowledge thatimpurities present in alginate polymers have been shown to limit thebiocompatibility of implanted alginates, ultra-pure, low viscosityalginate (UPLVG, FMC Biopolymers) was selected as a starting materialfor modification experiments.

Alkynes Used as Reagents for 1,3-Dipolar Cycloaddition

Alcohols Used as Reagents for Esterification

Amines used as Reagents for Amidation

Amines Used as Reagents to Introduce Azide Moieties

Unmodified alginate polymer was covalently modified by reaction withone, two, or three the esters, amines, and/or alkynes shown above in acombinatorial fashion. FIG. 1 shows the general structure of themodified alginates obtained using this method.

2. Representative Reaction Conditions

Due to the parallel and combinatorial nature of the modificationprocess, synthetic reactions were performed using a robotic core module.UPLVG alginate was selected as a starting material for modificationexperiments. In the first combinatorial reaction, the unmodifiedalginate was reacted with one of the alcohols, amines, and amines usedto introduce an azide moiety in the presence of2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methyl morpholine(NMM). In a second combinatorial step, each of the modified alginatepolymers formed above was reacted with another of the alcohols, amines,or amines used to introduce an azide moiety in the presence of2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methyl morpholine(NMM). In a final combinatorial step, all members of the library whichwere reacted with an amine used to introduce an azide moiety werefurther functionalized using a 1,3-dipolar cycloaddition reaction. Thosemembers of the library which had been reacted with 4-iodobenzylaminewere first reacted with sodium azide to convert the iodide moieties toazide moieties. Subsequently, all members of the library which werereacted with an amine used to introduce an azide moiety were reactedwith one of the alkynes used as reagents for 1,3-dipolar cycloadditionin the presence of CuSO₄/sodium ascorbate.

To optimize the biocompatibility of the chemically modified alginates, arigorous purification methodology was developed to eliminate potentiallyirritating impurities. Following each covalent modification, themodified alginates were filtered through a cyano-modified silica columnaimed at capturing bulk organic impurities. Finally, after completingall covalent modification steps, the modified alginates were dialyzedagainst 10,000 MWCO membrane to remove any remaining small-molecule orlow molecular weight impurities.

The purity of the modified alginates was determined by ¹H NMR analysis.The ¹H NMR spectra of each modified alginate polymer was collected, andpeaks corresponding to the modified alginate polymer and to anyimpurities were integrated to determine the relative quantity of eachspecies in the sample.

Example 2: High Throughput Screening of Modified Alginates Using aHydrogel Formation Assay

Covalent modification of the alginates affects the physical propertiesof the alginate, including the ability of the alginate to form hydrogelssuitable for the encapsulation of cells and biomolecules. To eliminatemodified alginates that have lost their ability to form hydrogels and tofurther focus our screening efforts on stronger candidates, afluorescence-based crosslinking assay was used to quantify the abilityof modified alginates to form hydrogels.

The hydrogel formation assay described herein exploits the ability ofhydrogels to trap fluorescent compounds, and differentially retain thefluorophores upon washing based on the stability of the hydrogel. Eachof the modified alginates prepared using the combinatorial approachdescribed in Example 1 was dissolved in water. A rhodamine dye thatfluoresces at 580 nm was added to each sample. The modified alginatesample was then crosslinked by the addition of a barium or calcium salt,for example BaCl₂, to induce formation of a hydrogel. After incubationfor 10 minutes, each sample was washed repeatedly with water. Thefluorescence intensity of each sample was measured using a fluorimeter.

Each modified alginate was screened three times, and the resultsobtained in the three trials were averaged. The average fluorescenceintensity values for each modified alginate were compared to thefluorescence levels of the negative control (water) and unmodifiedalginate (UPLVG). Modified alginates yielding fluorescence values belowthe negative control were considered unusable for applications wherehydrogel formation is critical (i.e. the encapsulation of cells).

Example 3: In Vitro Screening of Modified Alginates for Biocompatibility

The cytotoxicity of the modified alginate polymers on HeLa cells wasevaluated to screen for biocompatibility in vitro. The modifiedalginates identified in Example 2 as capable of forming hydrogels wereloaded into wells of 96-well plates which were coated withpoly-L-lysine. Unmodified alginate and saline were also loaded intowells of 96-well plates which were coated with poly-L-lysine ascontrols. A 100 mM BaCl₂ crosslinking solution was dispensed in all ofthe wells of the 96-well plate. The excess crosslinker was thenaspirated. HeLa cells were seeded into the wells and incubated for 3days at 37° C. in a humidified chamber.

A cell viability assay using3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasperformed, in which the media was aspirated from all wells and 100 μl ofDMEM media without phenol red and 10 μl MTT (5 mg/ml) were added to allof the wells of the 96-well plate. The plate was incubated for 4 hoursat 37° C. in a humidified chamber. After incubation, 85 μl of solutionwas aspirated and 100 μl DMSO was added. Purple formazan crystals formduring the assay in proportion to the number of viable HeLa cellspresent in each well. The contents of each well were pipetted up anddown to solubilize the formazan crystals prior to measurement. The platewas incubated at 37° C. for 10 minutes after which the bubbles fromagitation were removed. The plate was read using a UV/Vis plate readerat 540 nm with a reference at 700 nm. The viability was normalized tocells seeded in wells with no alginate.

The results of the cell viability are shown in FIG. 3, which plots theeffect of selected modified alginates on HeLa cell line viability ascompared to the positive control (no alginate). Alginate (Alg) has aviability of 53%. The assay identified modified alginate polymers whichdisplayed decreased cytotoxicity relative to unmodified alginate. Thesewere selected for further analysis.

Example 4: High Throughput In Vivo Screening of Modified Alginates toAssess Biocompatibility

Current biocompatibility analysis methods are slow and requirehistological analysis. In order to screen the large numbers of modifiedalginates prepared using the combinatorial synthetic methods describedherein, a high throughput in vivo biocompatibility assay was used toassess the relative biocompatibility of alginate polymers.

1. High Throughput In Vivo Screening Protocol

8-12 week old male SKH1 mice were obtained from Charles RiverLaboratories (Wilmington, Mass.). The mice were maintained at the animalfacilities of Massachusetts Institute of Technology, accredited by theAmerican Association of Laboratory Animal care, and were housed understandard conditions with a 12-hour light/dark cycle. Both water and foodwere provided ad libitum.

Injections were performed in accordance with ISO 10993-6: 2001. Prior toinjection all materials were sterilized via 0.22 μm filtration. The micewere anesthetized via isoflurane inhalation at a concentration of 1-4%isoflurane/balance O₂ to minimize movement. Their backs were scrubbedwith 70% isopropyl alcohol and the animals were injected with modifiedalginates in an array format on the animals' back for high-throughputscreening. Six injections were made in each mouse with one of theinjections being an unmodified alginate control. Injection volumes were100 μl.

On days 1, 3, 7, 14, 21, and 28 post-injection, host cell activity inresponse to the implantation of modified alginates was imagedkinetically using fluorescent whole animal imaging. 24 hours before invivo fluorescence imaging, 2 nmol of ProSense-680 (VisEn Medical,Woburn, Mass., excitation wavelength 680±10 nm, emission 700±10 nm)dissolved in 150 μl sterile PBS was injected into the tail vein of eachmouse to image cathepsin activity.

In vivo fluorescence imaging was performed with an IVIS-Spectrummeasurement system (Xenogen, Hopkinton, Mass.). The animals weremaintained under inhaled anesthesia using 1-4% isoflurane in 100% oxygenat a flow rate of 2.5 L/min. A binning of 8×8 and a field of view of13.1 cm were used for imaging. Exposure time and f/stop—the relativesize of the opening of the aperture—were optimized for each acquiredimage. Data were acquired and analyzed using the manufacturer'sproprietary Living Image 3.1 software. All images are presented influorescence efficiency, which is defined as the ratio of the collectedfluorescent intensity to an internal standard of incident intensity atthe selected imaging configuration. Regions of interest (ROIs) weredesignated around the site of each injection.

Biocompatibility of the materials was examined 14 days post injection.The fluorescence intensity measured at the implantation site of modifiedalginates was compared with the fluorescence intensity measured at theimplantation site of and unmodified alginates. Modified alginatesexhibiting smaller fluorescence intensity at the implantation site thanthe fluorescence intensity measured at the implantation site ofunmodified alginates were characterized as biocompatible. Modifiedalginates exhibiting greater fluorescence intensity at the implantationsite than the fluorescence intensity measured at the implantation siteof unmodified alginates were characterized as not biocompatible.

The in vivo stability of the alginate gels was assessed at 28 days postinjection. Modified alginates which remained at the site of injectionafter 28 days were characterized as capable of forming mechanicallystable hydrogels in vivo. Modified alginates which were not present atthe site of injection after 28 days were deemed to not capable offorming mechanically stable hydrogels in vivo, and were classified as‘failures’.

Modified alginates characterized as both biocompatible and capable offorming mechanically stable hydrogels in vivo were identified as ‘hits’,and selected for further study.

2. Validation of the High Throughput In Vivo Screening Protocol

In order to validate the high throughput in vivo screening assaydescribed above, modified alginates were subcutaneously injected intomice in an array format and crosslinked in situ as described above. Micewere imaged on days 1, 3, 7, 14, 21, and 28 post-injection usingfluorescent, whole animal imaging, and tissue samples were collectedafter imaging for histological analysis. To obtain tissue samples forhistological analysis, mice were euthanized via CO₂ asphyxiation and theinjected biomaterial and surrounding tissue were excised. The tissueswere then fixed in 10% formalin, embedded in paraffin, cut into 5 μmsections, and stained using hematoxylin and eosin (H&E) for histologicalanalysis by a board certified pathologist.

Fibrosis was rated on a scale where a zero involved no fibrosis, a oneindicated partial coverage with one to two layers of fibrosis, a two isdesignated a thicker fibrotic layer that nearly covered the implant, anda three denoted concentric fibrotic coverage of the polymer. Bothpolymorphonuclear (PMN) cells and macrophages were rated on a scalewhere no observed cells were indicated with a zero, scattered cellsscored a one, numerous cells clustering on the sides of the polymerscored a two, and numerous cells surrounding the material resulted in athree. Both the histological score and fluorescence response normalizedto alginate were examined for the whole library and materials thatoutperformed unmodified alginate were judged to be biocompatible. Thiscorresponds to a normalized fluorescent signal of <100% and a fibrosisscore of <3.

Data captured using whole animal imaging was demonstrated to displayedsimilar temporal trends in cellular recruitment of phagocytes to thebiomaterials compared to histological analysis. Accordingly, the highthroughput in vivo screening method described above was validated.

Example 5: In Vivo Screening of Modified Alginates to Quantify

Biocompatibility

8-12 week old male SKH1 mice were obtained from Charles RiverLaboratories (Wilmington, Mass.). The mice were maintained at the animalfacilities of Massachusetts Institute of Technology, accredited by theAmerican Association of Laboratory Animal care, and were housed understandard conditions with a 12-hour light/dark cycle. Both water and foodwere provided ad libitum.

Injections were performed in accordance with ISO 10993-6: 2001. Prior toinjection all materials were sterilized via 0.22 m filtration. The micewere anesthetized via isoflurane inhalation at a concentration of 1-4%isoflurane/balance O₂ to minimize movement. Their backs were scrubbedwith 70% isopropyl alcohol and the animals were injected with a modifiedalginate. The injection volume was 100 μl.

Cathepsin activity was measured 7 days post injection using an in vivofluorescence assay to quantify the foreign body response to the modifiedalginate. 24 hours before in vivo fluorescence imaging, 2 nmol ofProSense-680 (VisEn Medical, Woburn, Mass., excitation wavelength 680±10nm, emission 700±10 nm) dissolved in 150 μl sterile PBS was injectedinto the tail vein of each mouse to image cathepsin activity.

In vivo fluorescence imaging was performed with an IVIS-Spectrummeasurement system (Xenogen, Hopkinton, Mass.). The animals weremaintained under inhaled anesthesia using 1-4% isoflurane in 100% oxygenat a flow rate of 2.5 L/min. A binning of 8×8 and a field of view of13.1 cm were used for imaging. Exposure time and f/stop—the relativesize of the opening of the aperture—were optimized for each acquiredimage. Data were acquired and analyzed using the manufacturer'sproprietary Living Image 3.1 software. All images are presented influorescence efficiency, which is defined as the ratio of the collectedfluorescent intensity to an internal standard of incident intensity atthe selected imaging configuration. Regions of interest (ROIs) weredesignated around the site of each injection.

Fluorescence images were captured 7 days post-injection illustratingrelative cathepsin activity at the point of injection of selectedmodified alginates. The fluorescence intensity was measured andnormalized to the fluorescence response measured using unmodifiedalginate in order to quantify the biocompatibility of the modifiedalginates. The results obtained for selected modified alginates areincluded in FIG. 4.

Example 6: Treatment of Diabetes in STZ-Induced Diabetic Mice

The transplantation of biocompatible alginate-encapsulated beta cellsoffers potential as a treatment for diabetes. Pancreatic rat islet cellswere encapsulated using fourteen biocompatible modified alginatepolymers identified using the assays detailed above (includingPF_N287_B_B4, PF_N287_F2, PF_N287_G3, PF_N287_B3, PF_N287_B1,PF_N287_A4, PF_N287_B1, PF_N287_E3, PF_N263_C12, PF_N63_A12, PF_N287_E1,PF_N287_D3, PF_N263_A7, and PF_N263_C6). Alginate-encapsulated isletscapsules were fabricated from 750 μl of a 4% (w/v) solution of eachmodified alginate in deionized water containing suspended 1,000 isletssuspended using the Inotech encapsulator (Inotech) set to a voltage of1.05 kV, a vibration of 1225 Hz, and a flow rate of 10-25 ml/min with a300 μm nozzle. Alginate was crosslinked in a 20 mM BaCl₂ solution. Afterencapsulation, the capsules were washed twice with HEPES solution, fourtimes with Krebs solution, and twice with RPMI-1640 medium.

The encapsulated rat islet cells were transplanted into STZ induceddiabetic mice. Prior to transplantation, the mice were anesthetizedunder continuous flow of 1-4% isofluorane with oxygen at 0.5 L/min. Ashaver with size #40 clipper blade will be used to remove hair to revealan area of about 2 cm×2 cm on ventral midline of the animal abdomen. Theentire shaved area was aseptically prepared with a minimum of 3 cyclesof scrubbing with povidine, followed by rinsing with 70% alcohol. Afinal skin paint with povidine was also applied. The surgical site wasdraped with sterile disposable paper to exclude surrounding hair fromtouching the surgical site. A sharp surgical blade was used to cut a0.5-0.75 cm midline incision through the skin and the linea alba intothe abdomen. A sterile plastic pipette was used to transfer the alginatecapsules into the peritoneal cavity. The abdominal muscle was closed bysuturing with 5-0 Ethicon black silk or PDS-absorbable 5.0-6.0monofilament absorbable thread. The external skin layer was closed usingwound clips. These wound clips were removed 7-10 d post-surgery aftercomplete healing was confirmed.

Blood glucose levels in the STZ induced diabetic mice were monitoreddaily for between 20 and 30 days post-transplantation using a drop ofblood obtained by scrubbing the tail with 70% isopropyl alcohol andmaking a superficial cut on the skin of the tail to produce a drop ofblood. Mice were restrained during sampling in a rotating tail injector.

The blood glucose levels in the STZ induced diabetic mice followingislet transplantation are shown in FIG. 5. The dashed black linerepresents normoglycemia in mice. Pancreatic rat islet cells that wereencapsulated in modified alginates were able to reduce the blood glucoselevels in all cases, and in some cases, were even able to inducenormoglycemia.

Example 7: Particles Prepared from Mixture of Modified Alginate(s) andUnmodified Alginate

The growing recognition of the parameters driving fibrosis in vivo hasbeen applied to the analysis of the performance of modified alginates.Intraperitoneal (IP) implantation of modified alginate capsules revealedthat modified alginates may result in abnormally shaped capsules whencrosslinked using conditions defined for unmodified alginates. Theseabnormally shaped capsules can complicate implementation andinterpretation of modified alginate capsules implanted IP. In an effortto improve the capsule morphology, formulation methods for use withmodified alginate microparticles were developed where modified alginateswere blended with a small amount of high molecular weight alginate.Particles prepared from this mixture yielded particles with improvedmorphology and stability.

A 6% solution of modified alginate (w/w) was combined 1:1 by volume witha 1.15% solution of unmodified alginate (w/w). After mixing, capsulesare formed by following this solution through an electrostatic dropletgenerator, followed by crosslinking of the polymer in a 20 mM aqueousbarium chloride solution.

Particles prepared from modified alginate 263_A12 microparticlesformulated with barium and mannitol were compared to particles preparedfrom 263_A12 blended with a small amount of unmodified SLG100 alginate(16% by weight). The particles prepared from a mixture of modifiedalginate and unmodified alginate produced more homogenous microparticlepopulations. Quantitative fluorescence analysis with prosense at severaltime points with modified alginates blended with SLG100 was performed.The results are shown in FIG. 6. Several reformulated modified alginatesdisplayed less inflammatory response at day 7 compared to the controlalginate. Initial experiments with large capsules (1.5 mm diameter) showcomparably clean capsules after 2 weeks in the IP space ofimmunocompetent C57BL6 mice.

Data collected to date with these controlled capsules indicates thatreformulation and capsule morphology can have a significant effect oninflammation as measured by prosense. An improved inflammation responseis observed in some polymers (FIG. 6), while others are impactednegatively.

Example 8: Demonstration of Anti-Fibrotic Activity of Modified Alginates

In this example, a chemical modification approach was taken to mitigatethe immune recognition of alginate microspheres in preclinical fibrosismodels, including NHPs, which are relevant to translation in humans. Thelead materials evade immune recognition and fibrosis in the IP space ofboth C57BL/6 mice and cynomolgus macaques. This alginate blocksmacrophage adhesion, stunting activation of the foreign body responseand providing insight to the surface properties necessary to overcomethe fibrosis of implanted materials.

I. Methods

A. Alginate Chemical Modification

1. Alginate Amidation

Alginate (Pronova UPVLVG from NovaMatrix, 1 eq., 100 mg=0.52 mmol ofCOOH available for reaction) was dissolved as a 2% Alginate solution ina 3:2 water:acetonitrile mixture (5 ml total volume). Amine (N1 to N9,Z1, Z2) (1 eq, Sigma Aldrich or TCI America) was then added to themixture along with the coupling agent2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 0.5 eq., 45 mg, SigmaAldrich) and 4-Methylmorpholine (NMM, 1 eq., 56 μl, Sigma Aldrich). Themixture was stirred at 55° C. overnight and the solvent was removedunder reduced pressure. The resulting solid was dissolved in water andfiltered through cyano modified silica gel (Silicycle) to removeinsoluble precipitate. The resulting solution was then dialyzed againsta 10,000 MWCO dialysis membrane overnight with DI water to furtherpurify the polymer. The resulting solution was then lyophilized to getpurified compound.

2. Alginate Esterification

Alginate (Pronova UPVLVG from NovaMatrix, 1 eq., 100 mg=0.52 mmol ofCOOH available for reaction) was dissolved as a 2% Alginate solution ina 3:2 water:alcohol (O1 to O12) mixture (5 ml total volume). Thecoupling agent 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 0.5 eq., 45mg, Sigma Aldrich) and 4-Methylmorpholine (NMM, 1 eq., 56 μl, SigmaAldrich) was then added and the mixture was stirred at 55° C. overnight.The next day the solvent was removed under reduced pressure. Theresulting solid was dissolved in water and filtered through cyanomodified silica gel (Silicycle) to remove insoluble precipitate. Theresulting solution was then dialyzed against a 10,000 MWCO dialysismembrane overnight with DI water to further purify the polymer. Theresulting solution was then lyophilized to get purified compound.

3. Huisgen Cycloaddition (“Click”)

In a second step, alginates reacted with Z2 were dissolved in a solutionof water:methanol 1:1 (5 ml total). Sodium azide (0.25 eq., 19 mg, SigmaAldrich), sodium L-ascorbate (0.05 eq., 19 mg, Sigma Aldrich),trans-N,N′-Dimethylcyclohexane-1,2-diamine (0.25 eq., typically 20 μl,Sigma Aldrich), Copper(I)-Iodide (0.5 eq., 10 mg, Sigma Aldrich) wereadded as coupling agents. Then 0.51 mmol of the respective Alkyne (Y1 toY20) was added and the mixture was stirred at 55° C. overnight. Thesolvent was removed under reduced pressure. The resulting solid wasdissolved in water and filtered through cyano modified silica gel toremove insoluble precipitate. The clear solution was lyophilized anddissolved in 5 ml of water and dialyzed. The resulting solution was thendialyzed against a 10,000 MWCO dialysis membrane overnight with DI waterto further purify the polymer. The resulting solution was thenlyophilized to get purified compound.

In a second step, alginates reacted with Z1 were dissolved in a solutionof water:methanol 1:1 (5 ml total).Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 0.2 eq., 50 mg,Sigma Aldrich), Trietylamine (0.25 eq., typically 15 μl, Sigma Aldrich),Copper(I)-Iodide (0.25 eq., 5 mg, Sigma Aldrich) were added as couplingagents. Then 0.51 mmol of the respective Alkyne was added and themixture was stirred at 55° C. overnight. The solvent was removed underreduced pressure. The resulting solid was dissolved in water andfiltered through cyano modified silica gel to remove insolubleprecipitate. The clear solution was lyophilized, dissolved in 5 ml ofwater and dialyzed. The resulting solution was then dialyzed against a10,000 MWCO dialysis membrane overnight with DI water to further purifythe polymer. The resulting solution was then lyophilized to get purifiedcompound.

4. Optimized Syntheses for Preparation of Z2-Y12, Z1-Y15 and Z1-Y19:

Z2-Y12 Amine:

10 g of 2-(2-Propynyloxy) tetrahydopyran (1 eq. 71.36 mmol) was added toa solution of 5.1 g Sodium azide (1.1 eq, 78.5 mmol), 1.41 g Sodiumascorbate (0.1 eq, 7.14 mmol), 2.29 mlTrans-N—N′-Dimethylcyclohexane-1,2-diamine (0.25 eq, 17.83 mmol), 3.4 gCopper(I)-iodide (0.025 eq, 17.83 mmol) in 75 ml methanol. To thismixture 19.97 g of 4 Iodobenzylamide HCl was added. The reaction wasstirred overnight at 55° C. The solvent was removed under reducedpressure. The crude reaction was purified by liquid chromatography withdichloromethane:ultra (22% MeOH in DCM with 3% NH₄OH) mixture 0%→40% onsilica gel. The product was then reacted with alginate as describedbelow.

Z1-Y15 Amine:

3.5 g of 4-Propagylthiomorpholine 1,1-Dioxide (1 eq. 20 mmol) was addedto a solution of 2.5 g TBTA (0.2 eq, 4 mmol), 750 μl Triethylamine (0.5eq, 10 mmol), 250 mg Copper(I)-iodide (0.06 eq, 1.3 mmol) in 50 mlmethanol. The mixture was cooled to 0° C. and 5.25 ml of11-Azido-3,6,9-trioxaundecan-1-amine (1 eq, 20 mmol) was added. Thereaction was stirred overnight at 55° C. The solvent was removed underreduced pressure. The crude reaction was purified by liquidchromatography with dichloromethane:ultra (22% MeOH in DCM with 3%NH₄OH) mixture 0%->100% on a C18 column. The product was then reactedwith alginate as described below.

Z1-Y19 Amine:

3 g of 4-Ethynylaniline (1 eq. 20.2 mmol) was added to a solution of 2.5g TBTA (0.2 eq, 4 mmol), 750 μl Triethylamine (0.5 eq, 10.1 mmol), 250mg Copper(I)-iodide (0.06 eq, 1.31 mmol) in 50 ml methanol. The mixturewas cooled to 0° C. and 5.25 ml of 11-Azido-3,6,9-trioxaundecan-1-amine(1 eq, 20 mmol) was added. The reaction was stirred overnight at 55° C.The solvent was removed under reduced pressure. The crude reaction waspurified by liquid chromatography with dichloromethane:ultra (22% MeOHin DCM with 3% NH₄OH) mixture 0%→30% on a cyano functionalized silicacolumn. The product was then reacted with alginate as described below.

Alginate Reaction:

1.5 g of UPVLVG (1 eq) was dissolved in 45 ml of water and 675 mg of2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 0.5 eq) and 840 μl ofN-Methylmorpholine (NMM, 1 eq) was added. Then 7.65 mmol of the Z2-Y12,Z1-Y15, or Z1-Y19 amine was dissolved in 22.5 ml acetonitrile and addedto the mixture. The reaction was stirred overnight at 55° C. The solventwas removed under reduced pressure and the solid was dissolved in water.The solution was filtered through a pad of cyano functionalized silicaand the water was removed under reduced pressure to concentrate thesolution. It was then dialyzed against a 10,000 MWCO membrane in DIwater overnight. The water was removed under reduced pressure to givethe functionalized alginate.

B. Capsule Formation

An electrostatic droplet generator was set up as follows: an ES series0-100 KV, 20 Watt high voltage power generator (Gamma ES series, GammaHigh Voltage Research, FL, USA) is connected to the top and bottom of ablunt tipped needle (SAI Infusion Technologies, IL, USA). This needle isattached to a 5 mL lure lock syringe (BD, NJ, USA) which is clipped to asyringe pump (Pump 11 Pico Plus, Harvard Apparatus, MA, USA) that isoriented vertically. The syringe pump pumps alginate out into a glassdish containing a 20 mM barium 5% mannitol solution (Sigma Aldrich, MO,USA). The settings of the PicoPlus syringe pump are 12.06 mm diameterand 0.2 mL/min flow rate. After the capsules are formed, they are thencollected and then washed with hepes buffer (NaCl 15.428 g, KCl 0.70 g,MgCl2*6H2O 0.488 g, 50 mL of hepes (IM) buffer solution (Gibco, LifeTechnologies, California, USA) in 2 L of DiH2O) 4 times. The alginatecapsules are left overnight at 4° C. The capsules are then washed 2times in 0.8% saline and kept at 4° C. until use.

Solubilizing alginates: SLG20 (NovaMatrix, Sandvika, Norway) wasdissolved at 1.4% weight to volume in 0.8% saline. SLG100 (NovaMatrix,Sandvika, Norway) was dissolved at 1.2% weight to volume in 0.8% saline.UPVLVG (NovaMatrix, Sandvika, Norway) was dissolved at 5% weight tovolume in 0.8% saline. All modified alginates were initially dissolvedat 5% weight to volume in 0.8% saline. Modifies were then blended with3% weight to volume SLG100, dissolved in 0.8% saline (see Table 1 forratios).

Forming different sized capsules: for 300 μm diameter capsules, a 30gauge blunt tipped needle (SAI Infusion Technologies) was used with avoltage of 7-8 kV. For 500 μm diameter capsules, a 25 gauge blunt tippedneedle (SAI Infusion Technologies) was used with a voltage of 5-7 kV.For 1.5 mm capsules, an 18 gauge blunt tipped needle (SAI InfusionTechnologies) was used with a voltage of 5-7 kV.

TABLE 1 Modified Alginate to SLG100 blended volume ratios % Volume %Modified Modified Volume Alginate Solution SLG100 361_E9 70 30 411_RN880 20 411_OH6 60 40 411_OH9 60 40 411_OH11 50 50 411_OH3 80 20 411_RZA1580 20 411_RZA2 50 50 411_RZA19 70 30 411_RN7 80 20 VLVG/SLG100* 80 20Table 1 Legend: Modified alginates are blended with SLG100 to makecapsules. Modified alginate and SLG100 are drawn into a 5 mL syringe andthoroughly vortexed before encapsulation. Different modified alginatesrequire a different percent volume of SLG100 added to make sphericalcapsules. *VLVG/SLG100 is a control blend. The unmodified VLVG isblended with SLG100.

C. Transplantation of the Hydrogel Capsules and Other Material Spheres

All animal protocols were approved by the MIT Committee on Animal Care,and all surgical procedures and post-operative care was supervised byMIT Division of Comparative Medicine veterinary staff. Immune-competentmale C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) wereanesthetized with 3% isoflurane in oxygen and had their abdomens shavedand sterilized using betadine and isopropanol. A 0.5 mm incision wasmade along the midline of the abdomen and the peritoneal lining wasexposed using blunt dissection. The peritoneal wall was then graspedwith forceps and a 0.5-1 mm incision was made along the linea alba. Adesired volume of capsules were then loaded into a sterile pipette andtransplanted into the peritoneal cavity through the incision. Theincision was then closed using 5-0 taper tipped polydioxanone (PDS II)absorbable sutures. The skin was then closed over the incision using awound clip and tissue glue. Preoperatively, all mice also received a0.05 mg/kg dose of buprenorphine subcutaneously as a pre-surgicalanalgesic, along with 0.3 mL of 0.9% saline subcutaneously to preventdehydration.

D. Retrieval of Cells, Tissues, and Materials

At desired time points post-transplantation, as specified in figures andresults, mice were euthanized by CO₂ administration, followed bycervical dislocation. In certain instances, 5 ml of ice cold PBS wasfirst injected in order perform an intraperitoneal lavage to rinse outand collect free-floating intraperitoneal immune cells. An incision wasthen made using the forceps and scissors along the abdomen skin andperitoneal wall, and intraperitoneal lavage volumes were pipetted outinto fresh 15 ml falcon tubes (each prepared with 5 ml of RPMI cellculture media). Next, a wash bottle tip was inserted into the abdominalcavity. KREBS buffer was then used to wash out all material capsulesfrom the abdomen and into petri dishes for collection. After ensuringall the capsules were washed out or manually retrieved, if fibroseddirectly to intraperitoneal tissues, they were transferred into 50 mLconical tubes for downstream processing and imaging. Afterintraperitoneal lavage and capsule retrieval, remaining fibrosedintraperitoneal tissues were also excised for downstream FACS andexpression analyses.

E. Imaging of the Retrieved Material Capsules

For phase contrast imaging, retrieved materials were gently washed usingKrebs buffer and transferred into 35 mm petri dishes for phase contrastmicroscopy using an Evos XI microscope (Advanced Microscopy Group).

F. ProSense Assay

Female SKH1 mice (6 weeks old) were utilized for this assay. 100 ul ofcapsules were resuspended in 200 ul of saline, and injectedsubcutaneously into the mouse on the left side of upper back. The micewere feeded on AIN-93G purified rodent diet (TD 94045, Harlan) tominimize the fluorescent background after injection. Six days later, 100ul (4 nmol) of ProSence 750 FAST (NEV11171, PerkinElmer Inc.) per mousewas injected intravenously via tail vein. At day 7 (i.e., 24 hours postthe ProSense 750 FAST intravenous administration), the mice were scannedby IVIS Spectrum system (Xenogen, Caliper LifeScience). The mice wereanesthetized using 3% isofluorane in oxygen and maintained at the samerate throughout the procedure, and the settings of the IVIS Spectrumsystem were Exposure=7.50, Binning=Medium, FStop=2, Excitation=605 andEmission=660. The images were analyzed with LivingImage Software, andthe right side of upper back on the same mouse was used as controlduring the signal quantification.

G. Cell Staining and Confocal Immunofluorescence

Retrieved samples were stored in 4% paraformaldehyde overnight (dilutedin 1×PBS). Samples were then washed in Krebs Buffer (7.889 g NaCl, 0.35g KCl, 5.958 g HEPES (Sigma-Aldrich, Montana, USA), 0.163 g KH2PO4,0.144 g MGSO4*7H2O in 1000 mLs of DiH2O). Samples were washed with 10mLs of PBS. PBS was aspirated and 20 mL of 1% Triton X-100(Sigma-Aldrich, Montana, USA) solution was used to permeabilize cells.Samples were incubated for 10 minutes at room temperature. Samples werethen incubated in 15 mLs of 1% albumin solution (Sigma-Aldrich, Montana,USA), diluted in 1×PBS for 30 minutes at room temperature. 3 mLs ofantibody solution (1:200 CD68 488 Anti Mouse (BioLegend California,USA), 1:200 Anti-Mouse Actin, α-Smooth Muscle-Cy3(Sigma-Aldrich,Montana, USA), 1:30 Phalloidin anti mouse 647 (Life Technologies,California, USA), DAPI (NucBlue Live Cell Stain ReadyProbes, LifeTechnologies, California, USA) 2 drops per mL) all diluted in 1% albuminsolution was added to each sample. Samples were incubated in stainingsolution for 45 minutes at room temperature. Staining solution was thenaspirated. Samples were then washed twice with 20 mLs of 0.1% tween 20solution (Sigma-Aldrich, Montana, USA), diluted in 1×PBS. Samples werethen washed twice with 20 mLs of 1×PBS. Samples were then transferred toa 24 well glass bottom plate. Excess PBS was aspirated and 1 mL of 50%glycerol solution (Sigma-Aldrich, Montana, USA) was added. A Zeiss LSM700 system with ZEN microscope software was used to image and analyzethe stained samples. Obtained images where adjusted linearly forpresentation using Photoshop (Adobe Inc. Seattle, Wash.).

H. Protein Extraction

Cells on retrieved capsules were lysed by sonication for 30 seconds on30 seconds off cycle three times at 70% amplitude (QSonica Sonicator,Model #Q125, QSonica LLC) on ice in NP40 cell lysis buffer (Cat#FNN0021, Invitrogen) at the ratio of 100 ul capsules to 200 ul lysisbuffer, with 100 mM PMSF and 1× protease inhibitors (Halt Proteaseinhibitor single-use cocktail, Cat. #78430, Thermo Scientific). Lysateswere centrifuged for 20 min at 12000 rpm at 4° C.; the supernatant whichcontains proteins was aspirated in a fresh tube kept on ice. The pelletswere washed with the same volume of lysis buffer (i.e. the pellet of 100ul capsules were washed with 200 ul lysis buffer), and then centrifugedfor 20 min at 12000 rpm at 4° C., combined the supernatant with theprevious one. The proteins were stored at −80° C. for future use.

I. Elispot (Mouse Cytokine Array)

This assay was accomplished with Proteome Profiler Mouse Cytokine ArrayPanel A kit (Cat #ARY006, R&D system). For each membrane, 200 ul ofprotein solution was mixed with 100 ul of sample buffer (array buffer 4)and 1.2 ml of block buffer (array buffer 6), then added with 15 ul ofreconstituted Mouse Cytokine Array Panel A Detection Antibody Cocktailand incubated at room temperature for 1 hour. The array membrane wasincubated with block buffer (array buffer 6) for 2 hours on a rockingplatform shaker in the meantime, and then the block buffer wasaspirated, the prepared sample/antibody mixture was added onto themembrane and incubated overnight at 4° C. on a rocking platform shaker.The membrane was washed with 20 ml of 1× wash buffer for 10 minutes on arocking platform shaker for three time and rinsed with deionized wateronce, then was probed with Fluorophore-conjugated streptavidin (1:5,000dilution, Cat #926-32230, Li-Cor) at room temperature for 30 minutes ona rocking platform shaker, washed with wash buffer for three times andrinsed with deionized water once again as in above steps.Antibody-antigen complexes were visualized using Odyssey Detection(Li-Cor, Serial No. ODY-2329) at 800 nm wavelengths. The densities ofthe spots were analyzed by Image J software.

J. Western Blotting

12 ul of protein solution mixed with 1× loading buffer (SDS-Samplebuffer, Cat.#BP-111R, Boston BioProducts) for each lane was boiled at95° C. for 20 min and electrophoresed on SDS polyacrylamide gels (Any Kd15-well comb mini-gel, Biorad, Cat #456-9036), and 3 ul of PrecisionPlus Protein Dual Xtra Stands (Cat #161-0377, Bio-rad) was used asladder to indicate the position of the bands, and then blotted ontonitrocellulose membranes (Biorad, Cat. #162-0213). Blots were probedwith anti-αSmooth Muscle actin antibody (1:400 dilution, Rabbitpolyclonal to alpha smooth muscle Actin; Cat. #ab5694, AbCam) andanti-βactin antibody (1:4000 dilution, Monoclonal Anti-β-Actin antibodyproduced in mouse; Cat #A1978, Sigma Aldirch) as a loading controlfollowed by Donkey Anti-Rabbit (1 to 15,000 dilution, Cat #926-32213,Li-Cor) and Goat Anti-Mouse (1 to 15,000 dilution, Cat #926-68070,Li-Cor) Fluorophore-conjugated secondary antibodies. Antibody-antigencomplexes were visualized using Odyssey Detection (Li-Cor, Serial No.ODY-2329) at 700 and 800 nm wavelengths. The densities of the bands wereanalyzed by Image J software.

K. NanoString Analysis

RNAs for mock-transplanted (MT) controls, or for 500 or 1,500 μmalginate capsule-bearing mice (n=5/group) were isolated from tissuesamples taken at various time points after transplantation. RespectiveRNAs were quantified, diluted to the appropriate concentration (100ng/μl), and then 500 ng of each sample was processed according toNanoString manufacturer protocols for expression analysis via ourcustomized multiplexed 53-gene mouse macrophage subtyping panel. RNAlevels (absolute copy numbers) were obtained following nCounter(NanoString Technologies Inc., Seattle, Wash.) quantification, and groupsamples were analyzed using nSolver analysis software (NanoStringTechnologies Inc., Seattle, Wash.).

L. FACS Analysis

Single-cell suspensions of freshly excised tissues were prepared using agentleMACS Dissociator (Miltenyi Biotec, Auburn, Calif.) according tothe manufacturer's protocol. Single-cell suspensions were prepared inPEB dissociation buffer (1×PBS, pH 7.2, 0.5% BSA, and 2 mM EDTA) andsuspensions were passed through 70 μm filters (Cat. #22363548, FisherScientific, Pittsburgh, Pa.). All tissue and material sample-derived,single-cell populations were then subjected to red blood cell lysis with5 ml of 1×RBC lysis buffer (Cat. #00-4333, eBioscience, San Diego,Calif., USA) for 5 min at 4° C. The reaction was terminated by theaddition of 20 ml of sterile 1×PBS. The cells remaining were centrifugedat 300-400 g at 4° C. and resuspended in a minimal volume (˜50 μl) ofeBioscience Staining Buffer (cat. #00-4222) for antibody incubation. Allsamples were then co-stained in the dark for 25 min at 4° C. with two ofthe fluorescently tagged monoclonal antibodies specific for the cellmarkers CD68 (1 μl (0.5 μg) per sample; CD68-Alexa647, Clone FA-11, Cat.#11-5931, BioLegend), Ly-6G (Gr-1) (1 μl (0.5 μg) per sample;Ly-6G-Alexa-647, Clone RB6-8C5, Cat. #108418, BioLegend), CD11b (1 μl(0.2 μg) per sample; or CD11b-Alexa-488, Clone M1/70, Cat. #101217,BioLegend). Two ml of eBioscience Flow Cytometry Staining Buffer (cat.#00-4222, eBioscience) was then added, and the samples were centrifugedat 400-500 g for 5 min at 4° C. Supernatants were removed by aspiration,and this wash step was repeated two more times with staining buffer.Following the third wash, each sample was resuspended in 500 μl of FlowCytometry Staining Buffer and run through a 40 μm filter (Cat.#22363547, Fisher Scientific) for eventual FACS analysis using a BDFACSCalibur (cat. #342975), BD Biosciences, San Jose, Calif., USA). Forproper background and laser intensity settings, unstained, singleantibody, and IgG (labeled with either Alexa-488 or Alexa-647,BioLegend) controls were also run.

M. Intravital Imaging

For intravital imaging, SLG20 hydrogels of 500 μm and 1500 μm sizes wereloaded with Qdot 605 (Life technologies, Grand Island, N.Y.) andsurgically implanted intoC57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J mice as describedabove. After 7 days post transplantation, the mice were placed underisoflurane anesthesia and a small incision was made at the site of theoriginal surgery to expose beads. The mice were placed on an invertedmicroscope and imaged using a 25×, N.A. 1.05 objective on an OlympusFVB-1000 MP multiphoton microscope at an excitation wavelength of 860nm. Z-stacks of 200 μm (10 μm steps) were acquired at 2-minute intervalsfor time series of 20-45 minutes depending on the image. The mice werekept under constant isoflurane anesthesia and monitored throughout theimaging session. Obtained images were analyzed using Velocity 3D ImageAnalysis Software (Perkin Elmer, Waltham, Mass.).

N. Confocal Raman Spectroscopy

Sample Preparation:

A drop of hydrogel capsules with buffer solution was dried on the quartzcoverslip (043210-KJ, Alfa Aesar). In order to remove the salt fromdried buffer solution, a drop of distilled water was gently applied ontop of the dried sample and immediately absorbed by a tissue. By doingthat, dried hydrogel capsules are prepared for Raman mapping.

Instrumentation:

A custom-built NIR confocal Raman microscopy system was previouslyreported (Kang et al., Combined confocal Raman and quantitative phasemicroscopy system for biomedical diagnosis. Biomed. Opt. Exp.2(9):2484-2492 (2011); Kang et al., Measuring uptake dynamics ofmultiple identifiable carbon nanotube species via high-speed confocalRaman imaging of live cells. Nano Letters 12(12):6170-6174 (2012)).Briefly, a 785 nm wavelength Ti: Sapphire laser (3900S, Spectra-Physics)was used for sample excitation. The collimated beam was filtered by aband pass filter (BPF, LL01-785-12.5, Semrock) and redirected to thedual axes galvanometer mirrors. High-speed XY scanning was performed bythe galvanometer mirrors (CT-6210, Cambridge Technology). A 1.2 NA waterimmersion objective lens (Olympus UPLSAPO60XWIR 60X/1.20) was used toboth focus the laser light onto the sample and to collect theback-scattered light. A piezo actuator combined with a differentialmicrometer (DRV517, Thorlabs) was used to perform the coarse and fineadjustments, respectively, of the sample focus. A flip mirror was placedafter the tube lens so that the sample focal plane from the incoherenttransmission source can be observed using a video camera with 75×magnification. The back-scattered Raman light from the sample passesthrough two dichroic mirrors (DM1: Semrock LPD01-785RU-25, DM2: SemrockLPD01-785RU-25×36×1.1) and was collected by a multi-mode fiber (ThorlabsM14L01). The collected signal was delivered to the spectrograph(Holospec f/1.8i, Kaiser Optical Systems) and detected by athermoelectric-cooled, back-illuminated and deep depleted CCD (PIXIS:100BR_eXcelon, Princeton Instruments). LabView 8.6 software (NationalInstruments), data acquisition board (PCI-6251, National Instruments)and MATLAB 2013 software (Mathworks) were used to control the system,acquire the data, and analyze the data.

Raman Spectroscopy Measurement:

60 mW of 785 nm laser power was focused to a micron spot size and usedto raster scan the hydrogel samples. 30×30 spectra were acquired from 45μm×45 μm area with an integration time 1.0 s/pixel. The totalmeasurement time was approximately 15 minutes.

Data Processing:

Two Raman images are generated based on the intensities of two Ramanbands. These Raman images are resized and overlaid as red and greencolors on top of corresponding bright field image from the same area.

II. Polymer and Compound Characterization

N7: ¹H (400 MHz; D₂O): 3.10-4.10 (m, alginate protons), 4.20 (2H, s,H₂N—CH₂-Ph), 4.40-5.20 (m, alginate protons), 7.41 (2H, m, Phenyl), 7.49(3H, m, Phenyl)

IR (ATR): 3234, 1579, 1465, 1407, 1368, 1078, 810, 692, 517.

N8: ¹H (400 MHz; D₂O): 3.00-3.20 (m, alginate protons), 3.60 (8H, m,ethoxy), 3.60-5.10 (m, alginate protons).

IR (ATR): 3233, 2927, 2358, 1591, 1405, 1318, 1022, 945, 810.

O3: ¹H (400 MHz; D₂O): 1.90-2.10 (m, 4H, Furfuryl), 3.23 (m, 2H,Furfuryl) 3.26-4.00 (m, alginate protons), 4.03 (3H, m,O—CH2-C[furfuryl]), 4.10-5.20 (m, alginate protons)

IR (ATR): 3202, 3070, 2344, 1711, 1594, 1398, 1021, 715, 549

O6: ¹H (400 MHz; D₂O): 3.60-4.52 (m, alginate protons), 4.59 (2H, m,O—CH2-C[furfuryl]), 4.6-5.2 (m, alginate protons), 6.45 (2H, m,CH—CH═CH—O Furfuryl), 7.53 (1H, m, CH—CH═CH—O Furfuryl).

IR (ATR): 3232, 2360, 1614, 1410, 1028, 538.

O9: ¹H (400 MHz; D₂O): 0.20 (s, 9H, Furfuryl),) 3.10-5.20 (m, alginateprotons).

IR (ATR): 3310, 2939, 2360, 1592, 1406, 1316, 1081, 1020, 902, 770.

O11: ¹H (400 MHz; D₂O): 3.05-4.50 (m, alginate protons), 4.52 (2H, s,O—CH2-Ph), 4.52-5.2 (m, alginate protons), 6.88 (2H, m, Phenyl), 7.26(2H, m, Phenyl).

IR (ATR): 3370, 3089, 1597, 1517, 1454, 1235, 1207, 989, 835, 801, 561.

Z1-Y2: ¹H (400 MHz; D₂O): 3.05-3.40 (m, alginate protons), 3.40-3.66(16H, m, ethoxy), 3.75 (3H, s, methoxy) 3.8-5.1 (m, alginate protons),7.19 (1H, m, Phenyl), 7.50 (1H, m, Phenyl), 7.94 (1H, m, Phenyl), 8.00(1H, m, Phenyl), 8.49 (1H, s, triazole).

IR (ATR): 3144, 2922, 1592, 1400, 1329 1019, 943.

Z1-Y15: ¹H (400 MHz; D₂O): 3.07 (4H, s, N—CH₂—CH₂—S), 3.17-3.40 (m,alginate protons), 3.46 (4H, s, N—CH₂—CH₂—S), 3.50-3.70 (16H, m,ethoxy), 3.7-5.2 (m, alginate protons), 8.08 (1H, s, triazole).

IR (ATR):3268, 2933, 2250, 1602, 1409, 1292, 1119, 1023, 946.

Z1-Y19: ¹H (400 MHz; D₂O): 3.05-3.40 (m, alginate protons), 3.40-3.66(16H, m, ethoxy), 4.4-5.1 (m, alginate protons), 6.96 (2H, m, Phenyl),7.63 (3H, m, Phenyl), 8.23 (1H, s, triazole).

IR (ATR): 3234, 2929, 2361, 1593, 1406, 1317 1024, 947, 810.

Z2-Y12: ¹H (400 MHz; D₂O): 1.57-1.78 (m, 6H, pyran), 3.10-4.40 (m,alginate protons), 4.48 (4H, m, pyran), 4.50-5.10 (m, alginate protons),7.56 (2H, m, Phenyl), 7.76 (3H, m, Phenyl), 8.51 (1H, s, triazole).

IR (ATR): 3235, 2933, 2111, 1592, 1405, 1290, 1023, 946.

N4-N2: ¹H (400 MHz; D₂O): 2.72 (s, 3H, N—CH₃ Dioxolane) 2.77 (s, 3H,N—CH₃ Benzyl), 3.36 (2H, d, N—CH₂-Dioxolane), 3.55-4.20 (m, alginateprotons), 4.22 (2H, m, N—CH₂-Ph), 4.50-5.10 (m, alginate protons), 5.19(1H, m, CH2-CH—O Dioxolane), 7.51 (5H, m, Phenyl).

IR (ATR): 3250, 2894, 1601, 1409, 1127, 1088, 1029, 946.

O3-O10: ¹H (400 MHz; D₂O): 1.60-2.20 (m, 4H, Tetrahydrofurfuryl),3.55-5.10 (m, alginate protons), 3.78 (2H, m, CH₂—CH₂—OTetrahydrofurfuryl), 3.85 (3H, s, COO—CH₃), 4.13-4.30 (3H, m, N—CH₂—Tetrahydrofurfuryl).

IR (ATR): 3448, 2926, 2111, 1618, 1420, 1290, 1096, 948, 904.

N9-O8: ¹H (400 MHz; D₂O): 1.28 (m, 3H, N—CH₂—CH₃), 1.32 (m, 3H,O—CH₂—CH₃), 1.63 (m, 3H, N—CH₂—CH₂—CH₂—CH₂—OH) 1.74 (m, 3H,N—CH₂—CH₂—CH₂—CH₂—OH), 3.09-3.40 (m, 6H, CH₃—CH₂—N—CH₂—CH₂—CH₂—CH₂—OH),3.55-5.10 (m, alginate protons), 4.06 (m, 3H, O—CH₂—CH₃).

IR (ATR): 3422, 1709, 1655, 1611, 1474, 1395, 1042, 798.

Small Molecule Preparations:

¹H (400 MHz; MeOD): 1.57 (m, 4H, pyran), 1.72 (m, 1H, pyran), 1.82 (m,1H, pyran) 3.58 (m, 1H, pyran), 3.87 (s, 1H, NH₂—CH₂-Ph), 3.92 (m, 1H,pyran), 4.68 (d, 1H, J=12 Hz, O—CH₂-triazole), 4.79 (m, 1H, O—CH—Opyran), 4.97 (d, 1H, J=12 Hz, O—CH2-triazole), 7.54 (m, 2H, aromatic),7.80 (m, 2H, aromatic), 8.49 (s, 1H, triazole).

¹³C (400 MHz; MeOD): 20.3 (CH₂ pyran), 26.5 (CH₂ pyran), 31.5 (CH₂pyran), 46.1 (NH₂CH₂), 61.0 (O—CH₂—C), 63.3 (CH₂—O pyran), 99.5 (O—CH—Opyran), 121.6 (CH aromatic), 123.17 (CH triazole), 129.9 (CH aromatic),137.1 (Cq-N aromatic), 144.9 (Cq-C aromatic), 146.9 (C triazole).

High resolution MS: M+1=289.1665+3.1 ppm.

¹H (400 MHz; D₂O): 2.86 (2H, s, NH₂), 3.01 (4H, m, N—CH₂—CH₂—S), 3.10(4H, m, N—CH₂—CH₂—S), 3.55 (2H, t, J=5.2 Hz, NH₂—CH₂), 3.61 (8H, m, PEG)3.85 (2H, s, Thiomorpholine-CH₂-Triazole), 3.90 (2H, t, J=5.2 Hz,N—CH₂—CH₂—O), 4.59 (t, 2H, J=5.2, N—CH₂—CH₂—O), 7.99 (1H, s, triazole).

¹³C (400 MHz; MeOH): 41.7 (NH₂—CH₂), 51.42 (N—CH₂), 51.48 (N—CH₂Thiomorpholine) 52.1 (S—CH₂ Thiomorpholine) 52.4(Thiomorpholine-CH₂-Triazole), 70.4-72.1 (m, PEG), 126.0 (CH triazole),144.5 (C triazole).

High resolution MS: M+1=392.1968-6.1 ppm.

¹H (400 MHz; MeOD): 2.79 (t, 2H, J=5.2 Hz, NH₂—CH₂), 3.46 (t, 2H, J=5.2,NH₂—CH₂—CH₂), 3.53 (m, 4H, PEG), 3.61 (m, 4H, PEG), 3.91 (t, 2H, J=5.2,N—CH₂—CH₂—O), 4.58 (t, 2H, J=5.2, N—CH₂—CH₂—O) 6.76 (m, 2H, aromatic),7.54 (m, 2H, aromatic), 8.14 (s, 1H, triazole).

¹³C (400 MHz; MeOD): 41.6 (NH₂—CH₂), 51.4 (N—CH₂), 70.3-71.9 (m, PEG),116.4 (CH aromatic), 121.1 (Cq-C), 121.5 (CH triazole), 127.7 (CHaromatic), 149.4 (C—NH₂ aromatic), 149.5 (C triazole).

High resolution MS: M+1=336.2036-8.3 ppm.

III. Results

The amines and alcohols listed in Table 2 were used to prepare themodified alginates. In the first combinatorial reaction, UPLVG alginatewas reacted with one of the compounds in Table 2 in the presence of2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methyl morpholine(NMM). In order to prepare multiply modified alginates, the first stepwas repeated using a different alcohol or amine from Table 2. Alginatesmodified with amine Z2 were then reacted with sodium azide to preparethe corresponding azide-modified alginate. These alginates, along withalginates modified with amine Z1, were then reacted with one of thealkynes listed in Table 2 in the presence of CuSO₄ and sodium ascorbatein order to prepare tetrazole-modified alginates.

Following each covalent modification, the modified alginates werefiltered through a cyano-modified silica column to capture bulk organicimpurities. Finally, after completing all covalent modification steps,the modified alginates were dialyzed against 10,000 MWCO membrane toremove any remaining small-molecule or low molecular weight impurities.

The purity of the modified alginates was determined by ¹H NMR analysis.The ¹H NMR spectra of each modified alginate polymer was collected, andpeaks corresponding to the modified alginate polymer and to anyimpurities were integrated to determine the relative quantity of eachspecies in the sample.

TABLE 2 Chemical modifications of the 73 capsule formulations Alginate #Modifications 1 O1-O7 2 O3-O10 3 O1-O11 4 O9-O12 5 O3-O7 6 Z2-Y8 7Z1-Y20 8 SLG100 9 Z2-Y16 10 Z2-Y13 11 Z2-Y17 12 O5 13 O7-O9 14 Z2-Y15 15O8 16 O4-O1 17 Z2-Y4 18 N9-Z1-Y16 19 Z1-Y4 20 VLVG 21 Z1-Y6 22 Z2-Y11 23N1 24 N9 25 Z2-Y3 26 N6-Z1-Y18 27 O9-O2 28 Z2-Y2 29 Z1-Y7 30 O4-O7 31N4-N2 32 Z1-Y8 33 Z2-Y16 34 V/S 35 Z2 36 Z1-Y14 37 O9-O3 38 N2-Z2-Y6 39N2 40 O5-O9 41 Z2-Y15 42 Z1-Y18 43 O7 44 Z1-Y12 45 N9-Z1-Y18 46 O10 47Z2-Y7 48 Z1-Y10 49 N6 50 Z2-Y13 51 O12 52 N3 53 O4 54 Z1-Y11 55 Z1-Y1756 Z1-Y1 57 Z1-Y9 58 Z2-Y6 59 SLG20 60 N5 61 Z1-Y3 62 Z2-Y5 63 Z1 64 O665 Z1-Y15 66 Z1-Y2 67 N8 68 Z1-Y19 69 O3 70 Z2-Y12 71 N7 72 O9 73 O11

A. Chemical Modification of Alginate Curtails the Foreign Body Responsein C57BL/6 Mice

The physicochemical parameters governing anti-fibrotic properties arecurrently poorly understood, making rationally designed approacheschallenging (Williams, Biomaterials 29:2941-2953 (2008)). To betterunderstand which structural features are germane to anti-fibroticproperties, a pool of diverse chemical compounds was selected that canmodify latent functionalities and properties on the polymeric alginatebackbone (FIG. 7). A 774-membered alginate analogue library wasconstructed with a variety of amines, alcohols, azides, and alkynes(FIG. 9). Of the 774 alginate analogues, 35 analogues resulted inunacceptably low yields and 634 alginates were determined to be capableof gelation after modification. These alginates were then evaluated asbulk hydrogels in a subcutaneous high-throughput mouse to measure levelsof acute inflammation (Tables 3-7). 200 alginate analogues displayedlower levels of cathepsin activity than the control alginate UPVLVG, thealginate used as the starting material for the library synthesis.Component designations refer to the components of Table 2 and FIG. 7.This assay monitors neutrophil activation subcutaneously with an imagingagent which yields increased fluorescence in response to increasedneutrophil-mediated cathepsin activity. Two hundred analogues displayedfluorescent levels that were lower than the base unmodified, ultrapureVLVG alginate (FIG. 11).

Since microcapsules have been the preferred alginate geometry in bothdrug delivery and cell encapsulation applications, 70 of the top 200performing polymers (Table 2) from the initial screen were fabricatedinto 300 μm capsules and re-evaluated in the subcutaneous inflammationassay (FIG. 12). Using chemically-modified alginate proved problematicin constructing microspheres, and good capsule morphology was restoredby blending a small amount of ultrapure SLG100 alginate with thealginate analogue solution.

TABLE 3 Cathepsin Activity of Singularly Modified Alginate PolymersCathepsin Cathepsin Cathepsin Moiety Activity Moiety Activity MoietyActivity N8 3.69 Z2-Y16 1.37 Z1-Y4 0.48 N9 0.76 Z2-Y10 1.37 Z1-Y6 0.50N3 1.14 Z2-Y14 1.00 Z1-Y17 0.58 N6 1.37 Z2-Y5 1.14 Z1-Y1 0.82 N4 1.37Z2-Y12 0.84 Z1-Y2 0.48 N5 2040 Z2-Y7 1.37 Z1-Y11 0.64 N2 0.96 Z2-Y6 0.71Z1-Y10 0.54 N1 1.57 Z2-Y8 0.97 Z1-Y12 0.84 N7 0.39 Z2-Y20 1.57 Z1-Y130.76 O6 — Z2-Y3 1.37 Z1-Y16 0.92 O12 0.81 Z2-Y18 1.37 Z1-Y9 0.60 O11 —Z2-Y2 2.17 Z1-Y14 0.79 O5 1.17 Z2-Y4 1.60 Z1-Y15 0.82 O3 1.17 Z2-Y171.60 Z1-Y7 0.66 O4 0.96 Z2-Y1 1.77 Z1-Y5 0.42 O9 0.82 Z2-Y11 1.60 Z1-Y200.50 O7 0.72 Z2-Y9 0.8 Z1-Y3 0.42 O8 0.96 Z2-Y13 1.57 Z1-Y18 0.56 O20.97 Z2-Y15 0.91 Z1-Y19 0.54 O1 1.17 Z2-Y19 — Z1-Y8 0.58 O10 0.69

TABLE 4 Cathepsin Activity of Multiply Modified Alginate Polymers N8 N9N3 N6 N4 N5 N2 N1 N7 N7 — — 0.20 — — — — — — N9 0.51 — — — 0.42 0.540.92 — 1.37 N4 1.37 0.48 2.40 — −1.14  — 2.17 0.76 1.17 N2 — 0.81 — —0.86 0.80 −2.40  — — N6 — 0.60 — — — — — 0.91 1.37 N8 0.87 0.97 — — —1.17 0.86 — 0.97 O3 0.71 0.88 0.60 0.72 0.6  0.58 0.58 0.64 0.76 O2 0.630.99 0.78 0.69 0.91 0.64 0.75 0.72 1.17 O10 1.17 0.72 0.95 0.78 0.920.88 0.82 0.88 0.69 O1 0.97 0.91 0.80 0.76 0.92 0.89 0.88 0.88 0.75 O80.87 0.92 0.81 0.93 0.78 0.80 0.84 0.81 0.89 O12 0.76 0.88 0.79 0.870.81 0.75 0.82 0.75 0.76 O9 0.88 0.92 1.17 0.85 0.84 0.78 1.17 1.17 0.89O7 1.17 0.97 0.99 0.96 1.17 1.17 1.17 0.84 1.37 O4 0.89 0.71 1.17 0.781.37 1.14 0.76 0.91 1.60 O5 — 0.88 0.54 0.96 0.96 1.37 0.60 0.69 1.17O11 1.00 1.37 — 1.17 1.17 — 0.75 0.69 1.17 N1 2.57 1.77 — 1.17 — 0.56 —−0.50  1.17 N3 1.37 1.17 −0.82  0.75 — — 1.57 — — N5 0.75 1.60 0.69 1.60— — 0.63 — 0.24 O6 0.75 — 0.63 — 0.69 0.89 0.82 0.81 —

TABLE 5 Cathepsin Activity of Multiply Modified Alginate Polymers N2 N1N3 N4 N5 N7 N9 N8 N6 Z2-Y16 1.14 — 1.17 0.60 0.75 1.17 1.37 1.57 0.93Z2-Y10 — — 1.37 1.37 — 1.17 1.37 1.14 1.37 Z2-Y14 — — — — 1.60 1.97 1.771.77 1.77 Z2-Y5 1.37 1.17 0.96 1.37 0.87 0.93 0.86 0.66 0.66 Z2-Y12 1.371.77 1.37 1.37 1.97 1.17 1.14 0.96 0.88 Z2-Y7 0.75 0.66 0.99 0.76 0.81 —0.96 0.79 0.96 Z2-Y6 0.78 1.37 0.97 0.84 0.92 — 0.82 0.54 0.97 Z2-Y8 —1.17 — — 1.37 1.14 1.37 1.37 1.37 Z2-Y20 0.51 1.37 0.56 0.44 0.58 1.371.60 0.46 1.37 Z2-Y3 0.79 1.17 0.69 0.94 0.92 1.57 1.37 1.37 2.00 Z2-Y181.37 0.93 1.37 1.14 1.37 0.82 1.57 1.57 1.37 Z2-Y2 1.37 0.86 0.82 1.171.57 1.37 1.37 1.57 1.60 Z2-Y4 1.14 1.37 1.37 — 1.37 1.17 1.60 1.60 1.97Z2-Y17 1.37 — 1.37 1.17 0.94 1.37 1.00 1.17 1.37 Z2-Y1 1.17 — 1.37 0.92— 0.93 0.80 1.37 1.37 Z2-Y11 0.95 1.37 1.37 — — 1.37 — — — Z2-Y9 0.720.84 0.92 0.69 — 0.75 1.77 1.37 1.37 Z2-Y13 1.37 1.37 1.60 0.75 1.371.37 — 1.57 1.77 Z2-Y15 1.57 1.17 1.00 — — 1.57 — — — Z2-Y19 — — — — — —— — —

TABLE 6 Cathepsin Activity of Multiply Modified Alginate Polymers N1 N4N7 N3 N2 N8 N5 N9 N6 Z1-Y4 0.63 0.51 1.17 0.51 0.60 1.37 1.37 1.37 1.17Z1-Y6 1.17 0.88 1.17 0.84 0.75 0.87 0.75 1.57 0.99 Z1-Y17 0.72 0.78 0.910.58 0.79 0.56 0.76 0.81 0.69 Z1-Y1 0.63 0.58 1.17 0.50 0.54 0.50 0.501.17 1.17 Z1-Y2 1.00 1.37 1.17 1.37 1.37 1.37 1.37 1.37 1.37 Z1-Y11 1.001.37 1.37 1.37 1.37 1.00 1.17 1.34 1.37 Z1-Y10 1.14 1.00 1.37 1.37 1.141.37 1.37 0.97 1.37 Z1-Y12 1.37 1.17 1.37 1.37 1.37 1.17 1.37 1.17 1.17Z1-Y13 1.14 0.99 1.14 1.37 1.17 1.17 1.17 1.17 1.17 Z1-Y16 1.37 1.141.17 1.37 1.17 1.00 1.17 0.97 0.96 Z1-Y9 0.97 0.94 0.94 0.97 0.93 0.840.96 0.87 0.84 Z1-Y14 0.89 0.99 0.85 0.88 1.17 0.76 0.78 0.85 0.86Z1-Y15 0.94 0.92 0.99 0.89 1.17 1.17 1.37 0.89 1.37 Z1-Y7 1.37 0.77 1.000.76 1.37 0.76 0.75 1.14 1.17 Z1-Y5 1.14 0.99 1.17 0.96 1.00 1.37 1.571.17 1.17 Z1-Y20 0.75 0.84 1.14 0.97 1.14 1.37 1.17 0.81 0.79 Z1-Y3 1.371.17 0.86 1.17 1.37 1.37 0.94 0.58 0.96 Z1-Y18 0.75 0.82 — 0.77 — — —0.63 0.63 Z1-Y19 1.17 — 0.99 — — — — — 1.17 Z1-Y8 — 1.17 1.00 0.94 0.920.94 1.14 1.37 0.97

TABLE 7 Cathepsin Activity of Multiply Modified Alginate Polymers O6 O12O11 O5 O3 O4 O9 O7 O8 O2 O1 O10 O7 1.37 1.17 — 1.37 1.17 0.99 1.37 1.371.17 1.37 1.37 1.14 O9 1.14 1.37 — 1.37 1.37 1.37 1.17 1.17 1.37 1.141.77 1.14 O1 0.86 0.82 1.37 1.17 1.37 0.92 0.97 0.97 1.37 0.97 1.17 1.17O2 1.14 0.97 1.17 1.00 0.95 — 0.89 0.76 0.91 0.76 0.92 0.72 O6 1.37 — —0.90 — 0.99 1.37 1.17 0.85 0.79 0.78 1.37 O12 0.99 1.77 0.89 1.37 1.371.17 0.75 0.96 — — — 1.37 O3 0.95 1.60 1.37 — — 1.17 0.87 0.80 1.14 —0.93 0.97 O5 — 1.97 — — — 1.00 — — 1.17 — 0.79 0.94 O11 — 1.17 0.95 — —— — — — 1.17 1.37 1.14 O4 — 1.37 — — — — — — 0.94 0.92 0.97 1.14 O10 —1.57 0.88 0.99 0.96 1.17 — 1.17 — 0.80 1.17 1.37 O8 — 1.57 — 0.71 0.720.85 0.80 0.92 1.37 0.69 1.37 1.14

All modified microcapsule formulations required this blending, capsulesmade from a blended solution of unmodified alginates VLVG and SLG100(V/S) and the conventional SLG20 capsule formulation served as controls.Of the 70 formulated alginate microcapsules, an improved inflammationresponse was observed with several polymers (FIG. 12). The top 10modified microcapsules displayed inflammation levels of 10-40% lowerthan the control alginates. To see if these lower levels of acuteinflammation translated into lower levels of fibrosis, the implant sitesof these top 10 alginates were sampled, sectioned, and processedhistologically after 28 days. Three modified alginates, Z2-Y12, Z1-Y15,and Z1-Y19, displayed minimal fibrotic overgrowth with capsules able tofully detach from the surrounding tissue.

To test if the subcutaneous results translate into other implantationsites, 300 μm capsules of the top 10 lead modified alginates wereimplanted in the intraperitoneal (IP) space of C57BL/6 mice. Capsuleswere retrieved after 14 days and evaluated for the accumulation ofcellular and fibrotic tissue. Phase contrast imaging of retrievedcontrol capsules show a robust fibrotic response, with a white fibrouscollagenous deposition observed on the capsules with brown capsuleclumping. By comparison, the top ten lead modified alginates showvarying degrees of fibrosis, with the modified alginates Z2-Y12, Z1-Y15,and Z1-Y19 showing almost no fibrous deposition and emerging asmaterials with anti-fibrotic properties.

Cellular staining and confocal microscopy of the Z2-Y12, Z1-Y15, andZ1-Y19 capsules showed little to no presence of macrophages (CD68),myofibroblasts (SMA) or general cellular deposition (DAPI). Theconventional microcapsule alginate, however, showed significantquantities of these cell populations on the retrieved capsules. Cellulardeposition is a key mechanistic component of material recognition and aninitiator of collagenous deposition, and the absence of cells on thecapsule surface is a further illustration of the anti-fibroticproperties of these modified alginates. To confirm these results, theSMA levels of Z2-Y12, Z1-Y15, Z1-Y19, and the control capsulesquantified by Western Blotting. 40 different cytokines from proteinsamples extracted from the retrieved microcapsules were also profiled.The cytokine profile of Z2-Y12 microcapsules show the lowest cytokinelevels of all tested samples, indicative of an overall lowerinflammatory response. Importantly, retrieved Z2-Y12 capsules had lowprotein levels of TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, andCCL4 which are known mediators of the foreign body response and fibrosis(Rodriguez et al., J. Biomed. Mater. Res. A 89:152-159 (2009)).Quantification of seventy nine RNA sequences of known inflammationfactors and immune cell markers isolated from retrieved capsules alsosupport lower levels of inflammation for Z2-Y12 implants. The RNAprofile in the surrounding IP fluid and fat tissue of Z2-Y12 implantedmice also more closely resembled mock treatment than mice implanted withcontrol capsules, further demonstrating the lower inflammatory potentialof this material.

B. Anti-Fibrotic Alginates Show Lower Macrophage Adhesion

FACS analysis was performed on retrieved capsules after 14 days IP tocharacterize the different immune populations that are recruited toZ2-Y12 capsules compared to control capsules (FIG. 8). Z2-Y12 capsulesdisplayed significantly lower percentages of macrophage and neutrophilpopulations, suggesting that Z2-Y12 capsules may be interfering witheither the recruitment of immune cell populations or amplification of aninflammatory response. To see if lower macrophage recruitment wasevident in vivo, IP intra-vital imaging was performed 7 days afterimplantation of fluorescent Z2-Y12 capsules in MAFIA mice (wheremacrophages express GFP) and compared them to fluorescent SLG20capsules. SLG20 capsules show a large population of macrophages activelyaggregating at the surface of these capsules, an indication offoreign-body giant cell formation and a clear step towards fibrosis.Z2-Y12 capsules by comparison showed much lower levels of macrophagesnear the capsules and there was no visible macrophage aggregation.

The lack of immune cell recruitment/activation to the surface of Z2-Y12capsules is consistent with the chemical modification of the polymerchains creating differential surfaces. Confocal raman spectroscopicmapping was performed to determine the distribution of the Z2-Y12chemical modification in the microcapsule. Strikingly, the diagnosticraman signature for the tetrahydropyranyl modification had a higherintensity at the surface of the microcapsule than at the core.Freeze-fracture cryo-SEM was then performed on Z2-Y12 microcapsules toexamine both the surface and core topography of the alginate analoguemicrospheres and compare them to the control capsule formulations.Z2-Y12 capsules display a more variable porosity throughout the capsulecore compared to either the blended control or conventional SLG20capsules, with pores ranging from 1 μm to 10 μm in size. The surfacefeatures between the different capsule formulations are quite distinct;the surface of Z2-Y12 capsules show fewer cratered features. Thesesurface differences are likely created by interactions at the boundarylayer between the modified polymer chains and the surrounding aqueoussolution.

C. Z2-Y12, Z1-Y15, and Z1-Y19 Resist Fibrosis in Non-Human Primates

The lead materials, Z2-Y12, Z1-Y15, and Z1-Y19, were advanced intoprimate studies to test their anti-fibrotic properties in a NHP model.Previous reports from our lab have established that spheroid size isalso a key parameter in mitigating fibrosis, with larger spheres (>1 mm)displaying anti-fibrotic properties. 1.5 mm capsules of the conventionalSLG20 capsules and the new Z2-Y12, Z1-Y15, and Z1-Y19 formulatedcapsules were separately transplanted into non-human primates (n=3 each)using a minimally invasive laparoscopic procedure. Capsules wereretrieved by IP lavage at 2 and 4 weeks, with one primate from eachcohort allowed to continue for 6 months. By 14 days post-transplant,SLG20 1.5 mm capsules were also largely free and not embedded in tissueat 2 weeks as well. However, numerous capsules were fibrosed and clumpedtogether. The 1.5 mm Z2-Y12, Z1-Y15, and Z1-Y19 retained a highretrieval rate and minimal embedding into the surrounding tissue. Atboth 2 and 4 weeks Z2-Y12, Z1-Y15, and Z1-Y19 capsules displayedsignificantly reduced fibrotic responses in phase contrast imagingcompared to 1.5 mm SLG20 capsules. Confocal imaging and FACS analysis ofretrieved capsules showed large 1.5 mm SLG20 capsules had more extensiveimmune macrophage and fibrosis-associated activated myofibroblastcoverage, consistent with the visible fibrotic overgrowth seen in thephase contrast imaging.

To investigate whether increasing capsule size and/or the modifiedchemistries would maintain improved anti-fibrotic activity over a longerperiod of time, confocal staining was also performed on capsulesretrieved from NHPs after 6 months. SLG20 capsules showed significantand extensive fibrotic overgrowth, while Z2-Y12 capsules were stillclean, showing no associated macrophages or myofibroblasts. FACSanalysis displayed similar results with a lower macrophage compositionassociated with retrieved Z2-Y12 capsules.

The combination of both increased capsule size (1.5 mm) and modified(Z2-Y12) chemistry substantially improved biocompatibility even at the 6month time point. Large 1.5 mm Z2-Y12 capsules looked to have minimal(almost non-existent) levels of fibrosis throughout all time points,indicating that anti-fibrotic effects of large capsule size synergizewith those of modified Z2-Y12 chemistry.

The results in this example demonstrate that chemical modification ofone of the most widely used biomaterials, alginate, produces hydrogelsthat are able to resist foreign body reactions in both rodents andnon-human primates. The lead alginate analogues, Z2-Y12, Z1-Y15, andZ1-Y19, show minimal recognition by macrophages and other immune cells,low levels of inflammatory cytokines, and almost no visible fibrousdeposition in both rodents and non-human primates even after 6 months(Z2-Y12). The distribution of the Z2-Y12 chemical modification resultsin a unique hydrogel surface that inhibits macrophage adhesion,effectively mitigating the foreign body response to the biomaterial. Theresults show that chemical modification of existing biomaterials is aviable strategy to overcome their foreign body responses. These are thefirst biomaterials to resist the foreign body response in non-humanprimates and their versatility as alginate-based materials allows itsuse in multiple biomedical applications.

Example 9: Demonstration of Anti-Fibrotic Activity of Modified AlginatesEncapsulating Human Cells in Immunocompetent Animals

This example demonstrates the anti-fibrotic properties of the modifiedalginates encapsulating human cells, which are implanted in robustimmunocompetent STZ C57BL/6J mice for a long period of time. Theencapsulated human cells are actively secreting xenogeneic substancesincluding, but not limited to, proteins. The xenogeneic substancesshould elicit an intense immune response from the mice. Therefore, thistest represents a severe immune challenge to the implanted modifiedalginates. Nonetheless, the implanted modified alginates resist foreignbody responses and shield the encapsulated human cells from host foreignbody responses for long periods of time. As a result, the beneficialeffects of the human cells are exerted for long periods of time. Thisexample describes testing of modified alginates encapsulating humancells secreting insulin for their ability to reduce blood glucose levelsand maintain normoglycemia in STZ-induced diabetic mice. The implantsreduce blood glucose levels and maintain normoglycemia long-term in theSTZ C57BL/6J mice without the need for immune suppressants.

Diabetes is a global epidemic afflicting over 300 million people (Shawet al., Diabetes Res. Clin. Pract. 87:4-14 (2010)). While a rigorousregimen of blood glucose monitoring coupled with daily injections ofexogenous insulin remains the leading treatment for type one diabetics,patients still suffer ill effects due to the challenges associated withdaily compliance (Pickup et al., N. Engl. J. Med. 366:1616-1624 (2012)).In addition, the regulation of insulin secretion by the beta cells ofthe pancreatic islets of Langerhans in response to blood glucose levelis a highly dynamic process, which is imperfectly simulated by periodicinsulin injections (Robertson et al., N. Engl. J. Med. 350:694-705(2004)). The transplantation of donor tissue, either in the form of apancreas transplantation or infusion of cadaveric islets, are currentlyimplemented clinically as one strategy to achieve insulin independencefor type 1 diabetics (Shapiro et al., N. Engl. J. Med. 355:1318-1330(2006); Shapiro et al., N. Engl. J. Med. 343:230-238 (2000); Qi et al.,Acta Diabetologica 51:833-843 (2014)). This approach has been limiteddue to two major drawbacks: (1) the limited supply of available donortissue, and (2) the adverse effects associated with a lifetime ofimmunosuppression (Hirshberg et al., Current Diabetes Reports 7:301-303(2007); Gibly et al., Diabetologia, 54:2494-2505 (2011); O'Sullivan etal., Endocrine Reviews 32:827-844 (2011)). Methods to relieve the needfor life long immunosuppression must be developed to allow for thebroadest clinical implementation (Hirshberg et al., Current DiabetesReports 7:301-303 (2007); Shapiro et al., The Review of DiabeticStudies: RDS 9:385-406 (2012); Vogel et al., Diabetologia 56:1605-1614(2013)).

Cell encapsulation is a promising technology that overcomes the need ofimmunosuppression by protecting therapeutic tissues from host rejection(Dolgin, Nat. Med. 20:9-11 (2014); Jacobs-Tulleneers-Thevissen et al.,Diabetologia 56:1605-1614 (2013)). The most commonly investigated methodfor islet encapsulation therapy is the formulation of isolated isletsinto alginate microspheres (Jacobs-Tulleneers-Thevissen et al.,Diabetologia 56:1605-1614 (2013); Scharp et al., Advanced Drug DeliveryReviews 67-68:35-73 (2014)). Clinical evaluation of this technology indiabetic patients with cadaveric human islets has only achieved glycemiccorrection for short periods (Jacobs-Tulleneers-Thevissen et al.,Diabetologia 56:1605-1614 (2013); Basta et al., Diabetes Care34:2406-2409 (2011); Calafiore et al., Diabetes Care 29:137-138,(2006)). Implants from these studies are characterized by strongimmune-mediated foreign body responses that result in fibroticdeposition, nutrient isolation, and donor tissue necrosis (de Groot etal., Journal of Surgical Research 121:141-150 (2004); Tuch et al.,Diabetes Care 32:1887-1889 (2009)). Similar results are observed withencapsulated xenogeneic islets and pancreatic progenitor cells inpreclinical diabetic mouse or non-human primate models, where both thetherapeutic efficacy (Hirshberg et al., Current Diabetes Reports7:301-303 (2007)) of encapsulated cadaveric human islets and pig isletsis hampered by immunological responses (Elliot et al., TransplantationProceedings 37:3505-3508 (2005); Omer et al., Diabetes 52:69-75 (2003);Schneider et al., Diabetes 54:687-693 (2005)).

A major contributor to the performance of encapsulated cell implants isthe immune response to the biomaterials used for cell encapsulation (Limet al., Science 210:908-910 (1980); Jacobs-Tulleneers-Thevissen et al.,Diabetologia 56:1605-1614 (2013); Scharp et al., Advanced Drug DeliveryReviews 67-68:35-73 (2014)). Immune-mediated foreign body responses toimplanted materials commonly lead to tissue capsule formation thatresults in implant failure (King et al., Journal of Biomedical MaterialsResearch 57:374-383 (2001)). When implanted into the intraperitonealspace of non-human primates or rodents with robust immune systems suchas C57BL/6J, (King et al., Journal of Biomedical Materials Research57:374-383 (2001); Dang et al., Biomaterials 32, 4464-4470 (2011))alginate microspheres elicit foreign body reactions and fibrosis (Kinget al., Journal of Biomedical Materials Research 57:374-383 (2001); Danget al., Biomaterials 32:4464-4470 (2011)).

A large library of chemically modified alginates was recently developedand evaluated for their potential to resist implant rejection in bothrodent and non-human primate models. This example extends that work toshow that triazole-thiomorpholine dioxide (TMTD; FIG. 13)alginate-encapsulating human cells were able to mitigate foreign bodyresponses in immune-competent C57BL/6 mice (FIGS. 14 through 16). As aresult, the TMTD alginate-encapsulating human cells are able to providelong-term glycemic correction and glucose-responsiveness. These resultsdemonstrate that these new materials can be used to provide long term,glycemic correction through implantation of microencapsulated humancell, thus improving therapeutic effect of such implanted cells.

This example demonstrates the successful use potential of encapsulatedhuman cells in immunocompetent animals for the restoration ofnormoglycemia without immune suppression. This shows the expectationthat the disclosed modified alginates can be used to encapsulate cellsand coat material and keep immune reactions to the cells and materialsat bay in subjects in which they are implanted. To ensure properbiocompatibility assessment in these studies an immunocompetentstreptozotocin-induced diabetic C57BL/6 mouse model was used becausethis strain is known to produce a strong fibrotic and foreign bodyresponse similar to observations made in human patients (Kolb et al., J.Respir. Cell. Mol. Biol. 27:141-150 (2002)). Formulations that haveshown glycemic correction utilizing other tissue sources, such asconventional microencapsulation with alginate (Lim et al., Science 210;908-910 (1980); Calafiore et al., Diabetes Care 29:137-138, (2006)) andlarger sphere formulations (Veiseh et al., Nat. Mater., DOI:10.1038/NMAT4290), were unable to support glycemic correction with humancells.

All materials were implanted intraperitoneally and retrieved atspecified times from immunocompetent streptozotocin induced diabeticC57BL/6 or B6.129S6-Ccr6tm1(EGFP)Irw/J mice in accordance with approvedprotocols and federal guidelines. Sample processing, staining, FACS, andimaging were performed as detailed in below.

I. Materials

All chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.) and cellculture reagents from Life Technologies (Grand Island, N.Y.), unlessotherwise noted. Antibodies: Alexa Fluor 488-conjugated anti-mouse CD68(Cat. #137012, Clone FA-11) and Alexa Fluor 647-conjugated anti-mouseLy-6G/Ly-6C (Gr-1) (Cat. #137012, Clone RB6-8C5) were purchased fromBioLegend Inc. (San Diego, Calif.). Cy3-conjugated anti-mouse alphasmooth muscle actin antibody was purchased from Sigma Aldrich (St. LouisMo.). Filamentous actin (F-actin)-specific Alexa Fluor 488-conjugatedPhalloidin was purchased from Life Technologies (Grand Island, N.Y.).Anti-Glucagon cat #ab82270, Anti-insulin cat #ab7842, Goat Anti-Guineapig IgG H&L conjugated Alexa Fluor® 488 cat #ab150185, and GoatAnti-Mouse IgG H&L conjugated Alexa Fluor® 594 cat #ab150116 werepurchased from abcam (Cambridge, Mass.). Anti-human C-peptide cat#GN-1D4 was purchased from Developmental Studies Hybridoma Bank(University of Iowa, Iowa City, Iowa). A sampling of the spheres used inthis study was submitted for endotoxin testing by a commercial vendor(Charles River, Wilmington, Mass.) and the results showed that spherescontained <0.05 EU/ml of endotoxin levels.

II. Methods

A. Fabrication of Alginate Hydrogel Capsules and Cell Encapsulation

All buffers were sterilized by autoclave and alginate solutions weresterilized by filtration through a 0.2 um filter. After solutions weresterilized, aseptic processing was implemented by performing capsuleformation in a type II class A2 biosafety cabinet to maintain sterilityof manufactured microcapsules/spheres for subsequent implantation. Thehydrogel capsules following the protocol described in Example 8.

To solubilize alginates, SLG20 (NovaMatrix, Sandvika, Norway) wasdissolved at 1.4% weight to volume in 0.8% saline. TMTD alginate wasinitially dissolved at 5% weight to volume in 0.8% saline, and thenblended with 3% weight to volume SLG100 (also dissolved in 0.8% saline)at a volume ratio of 80% TMTD alginate to 20% SLG100.

For formation of 0.5 mm spheres were generated with a 25 G blunt needle,a voltage of 5 kV and a 200 μl/min flow rate. For formation of 1.5 mmspheres, an 18 gauge blunt tipped needle (SAI Infusion Technologies) wasused with a voltage of 5-7 kV.

Cultured human cells were used for encapsulation. Immediately prior toencapsulation, the cultured human cell clusters were centrifuged at1,400 rpm for 1 minute and washed with Ca-free Krebs-Henseleit (KH)Buffer (4.7 mM KCl, 25 mM HEPES, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄×7H₂O, 135mM NaCl, pH≈7.4, ≈290 mOsm). After washing, the human cells werecentrifuged again and all supernatant was aspirated. The human cellpellet was then re-suspended in the SLG20 or TMTD alginate solutions(described above) at cluster densities of 1,000, 250, and 100 clustersper 0.5 ml alginate solution. Spheres were crosslinked using a BaCl₂gelling solution and their sizes were controlled as described above.Immediately after crosslinking, the encapsulated human cell clusterswere washed 4 times with 50 mL of CMRLM media and cultured overnight ina spinner flask at 37° C. prior to transplantation. Due to an inevitableloss of human cell clusters during the encapsulation process, the totalnumber of encapsulated clusters were recounted post-encapsulation.

B. Transplantation Surgeries

All animal protocols were approved by the MIT Committee on Animal Care,and all surgical procedures and post-operative care was supervised byMIT Division of Comparative Medicine veterinary staff. Immune-competentmale STZ-induced diabetic C57BL/6 mice (Jackson Laboratory, Bar Harbor,Me.) or male B6.129S6-Ccr6tm1(EGFP)Irw/J mice (Jackson Laboratory, BarHarbor, Me.) were anesthetized with 3% isoflurane in oxygen and hadtheir abdomens shaved and sterilized using betadine and isopropanol.Preoperatively, all mice also received a 0.05 mg/kg dose ofbuprenorphine subcutaneously as a pre-surgical analgesic, along with 0.3mL of 0.9% saline subcutaneously to prevent dehydration. A 0.5 mmincision was made along the midline of the abdomen and the peritoneallining was exposed using blunt dissection. The peritoneal wall was thengrasped with forceps and a 0.5-1 mm incision was made along the lineaalba. A desired volume of spheres (all materials without islets, as wellas SLG20 spheres encapsulating rat islets) were then loaded into asterile pipette and implanted into the peritoneal cavity through theincision. The incision was then closed using 5-0 taper-tippedpolydioxanone (PDS II) absorbable sutures. The skin was then closed overthe incision using a wound clip and tissue glue.

C. Blood Glucose Monitoring

To create insulin-dependent diabetic mice, healthy C57BL/6 mice weretreated with Streptozotocin (STZ) by the vendor (Jackson Laboratory, BarHarbor, Me.) prior to shipment to MIT. The blood glucose levels of allthe mice were retested prior to transplantation. Only mice whosenon-fasted blood glucose levels were above 400 mg/dL for two consecutivedays were considered diabetic and underwent transplantation.

Blood glucose levels were monitored three times a week followingtransplantation of islet-containing alginate capsules. A small drop ofblood was collected from the tail vein using a lancet and tested using acommercial glucometer (Clarity One, Clarity Diagnostic Test Group, BocaRaton, Fla.). Mice with unfasted blood glucose levels below 200 mg/dLwere considered normoglycemic. Monitoring continued until experimentaltime points had been reached, at which point they were euthanized andthe spheres were retrieved.

D. Human c-Peptide Monitoring

Human c-peptide levels were monitored every three weeks followingtransplantation of human cell-containing alginate capsules. Mice werefasted for 1 hour before blood collection, at which point approximately100-150 μL of blood was collected retro-orbitally into a serumcollection tube. Collected blood was centrifuged for 10 minutes at 13000rpm, serum was removed, and stored at 20° C. until assayed. Serum wasassayed for human c-peptide using the Alpco human c-peptide kit (Catalog#: 80-CPTHU-E10) according to the manufacturer's instructions.

E. Retrieval of Cells, Tissues and Materials

Retrieval of cells, tissues and materials was performed as describedabove in Example 8.

F. Imaging of the Retrieved Material Spheres

For phase contrast imaging of retrieved materials was performedfollowing the protocol described in Example 8.

For bright-field imaging of retrieved materials, samples were gentlywashed using Krebs buffer and transferred into 35 mm petri dishes forbright-field imaging using a Leica Stereoscopic microscope.

G. Confocal Immunofluorescence

Immunofluorescence imaging was used to determine immune populationsattached to spheres. Materials were retrieved from mice and fixedovernight using 4% paraformaldehyde at 4° C. Samples were then washedtwice with KREBS buffer, permeabilized for 30 min using a 0.1% TritonX100 solution, and subsequently blocked for 1 hour using a 1% bovineserum albumin (BSA) solution. Next, the spheres were incubated for 1hour in an immunostaining cocktail solution consisting of DAPI (500 nM),specific marker probes (1:200 dilution) in BSA. After staining, sphereswere washed three times with a 0.1% Tween 20 solution and maintained ina 50% glycerol solution. Spheres were then transferred to glass bottomdishes and imaged using an LSM 700 point scanning confocal microscope(Carl Zeiss Microscopy, Jena Germany) equipped with 5 and 10×objectives. Obtained images were adjusted linearly for presentationusing Photoshop (Adobe Inc. Seattle, Wash.).

H. Proteomic Analysis

1. Reduction, Alkylation and Tryptic Digestion

Retrieved samples were suspended in urea cell lysis buffer (8 M urea,Tris pH 8.0) and incubated at 4° C. overnight. Equivalent amounts ofprotein were reduced (10 mM dithiothreitol, 56° C. for 45 min) andalkylated (50 mM iodoacetamide, room temperature in the dark for 1 h).Proteins were subsequently digested with trypsin (sequencing grade,Promega, Madison, Wis.), at an enzyme/substrate ratio of 1:50, at roomtemperature overnight in 100 mM ammonium acetate pH 8.9. Trypsinactivity was quenched by adding formic acid to a final concentration of5%. Peptides were desalted using C18 SpinTips (Protea, Morgantown, W.Va.) then lyophilized and stored at −80° C.

2. TMT Labeling

Peptides were labeled with TMT 6plex (Thermo) per manufacturer'sinstructions. Lyophilized samples were dissolved in 70 μL ethanol and 30μl of 500 mM triethylammonium bicarbonate, pH 8.5, and the TMT reagentwas dissolved in 30 μl of anhydrous acetonitrile. The solutioncontaining peptides and TMT reagent was vortexed, incubated at roomtemperature for 1 h. Samples labeled with the six different isotopic TMTreagents were combined and concentrated to completion in a vacuumcentrifuge. For the first analysis samples were labeled using the TMT6plex channels as follows: 126—RZA 1.5 mm 250 biological replicate 1;127—RZA 1.5 mm 250 biological replicate 2; 128—SLG20 1.5 mm 250biological replicate 1; 129—SLG20 1.5 mm 250 biological replicate 2;130—SLG20 500 μm 250 biological replicate 1; and 131—SLG20 5001 μm 250biological replicate 2. For the second analysis samples were labeledusing the TMT 6plex channels as follows: 126—RZA 1.5 mm 250 biologicalreplicate 3; 127—RZA 1.5 mm 250 biological replicate 4; 128—SLG20 1.5 mm250 biological replicate 3; 129—SLG20 1.5 mm 250 biological replicate 4;130—SLG20 500 μm 250 biological replicate 3; and 131—SLG20 500 μm 250biological replicate 4.

3. LC-MS/MS

Peptides were then loaded on a precolumn and separated by reverse phaseHPLC (Thermo Easy nLC 1000) over a 140 minute gradient beforenanoelectrospray using a QExactive mass spectrometer (Thermo). The massspectrometer was operated in a data-dependent mode. The parameters forthe full scan MS were: resolution of 70,000 across 350-2000 m/z, AGC3e⁶, and maximum IT 50 ms. The full MS scan was followed by MS/MS forthe top 10 precursor ions in each cycle with a NCE of 32 and dynamicexclusion of 30 s. Raw mass spectral data files (.raw) were searchedusing Proteome Discoverer (Thermo) and Mascot version 2.4.1 (MatrixScience). Mascot search parameters were: 10 ppm mass tolerance forprecursor ions; 0.8 Dathe fragment ion mass tolerance; 2 missedcleavages of trypsin; fixed modification was carbamidomethylation ofcysteine; variable modification was methionine oxidation. TMTquantification was obtained using Proteome Discoverer and isotopicallycorrected per manufacturer's instructions.

I. Histological Processing for H&E and Masson's Trichrome Staining

Retrieved materials were fixed overnight using 4% paraformaldehyde at 4°C. After fixation, alginate sphere or retrieved tissue samples werewashed using 70% alcohol. The materials were then mixed with 4 degreescalcium-cooled Histogel (VWR, CA #60872-486). After the molds hardened,the blocks were processed for paraffin embedding, sectioning andstaining according to standard histological methods.

J. Histological Immunostaining

Paraffin embedded sectioned samples were stained for the following:human insulin (Anti-insulin cat #ab7842, abcam), human c-peptide(C-peptide cat #GN-1D4, Developmental Studies Hybridoma Bank, Universityof Iowa), human glucagon (Anti-Glucagon cat #ab82270, abcam). Cellularnuclei were stained with DAPI (cat #D1306, Life Technologies).

Paraffin slides were deparaffinized through subsequent incubations inthe following solvents (Xylene 5 min 2×100% ETOH 2 min×2 95% 2 min×2 70%2 min×2 d-water). Antigen retrieval was done by incubating sections for30 min in ice cooled PBS, and then blocking with 3% horse serum to blockfor 30 min. Antibody mixtures were then applied as follows: PrimaryA—Mix together Glucagon 1 to 200 and c-peptide 1 to 500. Primary B—Mixtogether Human insulin 1 to 500 and glucagon 1 to 200, incubate for 2hours and then Wash in PBS 10 min×4. Secondary A—Add anti-mouse AF594 1to 500 and anti-rat AF488 1 to 500. Secondary B—Add anti-guinea pigAF488 1 to 500 with anti-mouse AF594 1 to 500 incubate for 30 min thenwash 10 min 4×. Slides were then stained with DAPI and coverslipsmounted using prolong gold antifade (Life Technologies, Carlsbad,Calif.).

K. Western Blotting

Protein was extracted directly from materials for western blot analysis.For protein analyses, retrieved materials were prepared by immersingmaterials in Pierce RIPA buffer (Cat. #89901, Thermo Scientific) withprotease inhibitors (Halt Protease inhibitor single-use cocktail, Cat.#78430, Thermo Scientific) on ice, and then lysed by sonication (for 30seconds on, 30 seconds off, twice at 70% amplitude). Samples were thensubjected to constant agitation for 2 hours at 4° C. Lysates were thencentrifuged for 20 min at 12,000 rpm at 4° C., and protein-containingsupernatants were collected in fresh tubes kept on ice. In samples fromfat tissue, an excess of fat (a top layer on the supernatant) was firstremoved before supernatant transfer. 20 μg protein (quantified by BCAassay, Pierce BCA protein assay kit, Cat. #23225, Thermo Scientific) foreach lane was boiled at 95° C. for 5 min and electrophoresed onSDS-polyacrylamide gels (Any kD 15-well comb mini-gel, Biorad, Cat.#456-9036) and then blotted onto nitrocellulose membranes (Biorad, Cat.#162-0213). Blots were probed with anti-αSmooth Muscle actin antibody(1:400 dilution, Rabbit polyclonal to alpha smooth muscle actin; Cat.#ab5694, AbCam), anti-PDX1 antibody (1:1000 dilution, Rabbit polyclonalto pancreatic & duodenal homeobox 1; Cat. #06-1379, EMD Millipore), andanti-β-actin antibody (1:4000 dilution, monoclonal anti-β-actin antibodyproduced in mouse; Cat. #A1978, Sigma Aldrich) as a loading controlfollowed by donkey anti-rabbit (1:15,000 dilution, Cat. #926-32213,Li-Cor) and goat anti-mouse (1:15,000 dilution, Cat. #926-68070, Li-Cor)fluorophore-conjugated secondary antibodies. Antibody-antigen complexeswere visualized using Odyssey detection (Li-Cor, Serial No. ODY-2329) at700 and 800 nm wavelengths.

L. FACS Analysis

Single-cell suspensions of freshly excised tissues were prepared using agentleMACS Dissociator (Miltenyi Biotec, Auburn, Calif.) according tothe manufacturer's protocol. Single-cell suspensions were prepared in apassive PEB dissociation buffer (IX PBS, pH 7.2, 0.5% BSA, and 2 mMEDTA) and suspensions were passed through 70 μm filters (Cat. #22363548,Fisher Scientific, Pittsburgh, Pa.). This process removed the majorityof cells adhered to the surface (>90%). All tissue and materialsample-derived, single-cell populations were then subjected to red bloodcell lysis with 5 ml of 1×RBC lysis buffer (Cat. #00-4333, eBioscience,San Diego, Calif., USA) for 5 min at 4° C. The reaction was terminatedby the addition of 20 ml of sterile 1×PBS. The cells remaining werecentrifuged at 300-400 g at 4° C. and resuspended in a minimal volume(˜50 μl) of eBioscience Staining Buffer (cat. #00-4222) for antibodyincubation. All samples were then co-stained in the dark for 25 min at4° C. with two of the fluorescently tagged monoclonal antibodiesspecific for the cell markers CD68 (1 μl (0.5 μg) per sample;CD68-Alexa647, Clone FA-11, Cat. #11-5931, BioLegend), Ly-6G (Gr-1) (1μl (0.5 μg) per sample; Ly-6G-Alexa-647, Clone RB6-8C5, Cat. #108418,BioLegend), CD11b (1 μl (0.2 μg) per sample; or CD11b-Alexa-488, CloneM1/70, Cat. #101217, BioLegend), CD19 (1 μl (0.2 μg) per sample;CD19-Alexa-647, Clone HIB19, Cat. #302222, BioLegend), or IgM (1 μl (0.2μg) per sample; IgM-FITC, Clone RMM-1, Cat. #406505, BioLegend), CD8 (1μl (0.2 μg) per sample, BioLegend). Two ml of eBioscience Flow CytometryStaining Buffer (cat. #00-4222, eBioscience) was then added, and thesamples were centrifuged at 400-500 g for 5 min at 4° C. Supernatantswere removed by aspiration, and this wash step was repeated two moretimes with staining buffer. Following the third wash, each sample wasresuspended in 500 μl of Flow Cytometry Staining Buffer and run througha 40 μm filter (Cat. #22363547, Fisher Scientific) for eventual FACSanalysis using a BD FACSCalibur (cat. #342975), BD Biosciences, SanJose, Calif., USA). For proper background and laser intensity settings,unstained, single antibody, and IgG (labeled with either Alexa-488 orAlexa-647, BioLegend) controls were also run.

M. Intravital Imaging

For intravital imaging, human cell-containing hydrogels of 0.5 mm and1.5 mm sizes were fabricated with Qdot 605 (Life technologies, GrandIsland, N.Y.) and surgically implanted intoB6.129S6-Ccr6^(tm1(EGFP)Irw)/J mice as described above.

After 14 days post implantation, the mice were placed under isofluraneanesthesia and a small incision was made at the site of the originalsurgery to expose beads. The mice were placed on an inverted microscopeand imaged using a 25×, N.A. 1.05 objective on an Olympus FVB-1000 MPmultiphoton microscope at an excitation wavelength of 860 nm. Z-stacksof 200 μm (10 μm steps) were acquired at 2-minute intervals for timeseries of 20-45 minutes depending on the image. The mice were kept underconstant isoflurane anesthesia and monitored throughout the imagingsession. Obtained images were analyzed using Velocity 3D Image AnalysisSoftware (Perkin Elmer, Waltham, Mass.).

N. In Vivo Glucose Challenges (GSIS)

Mice were fasted overnight (12 hours) prior to glucose challenge. On theday of the challenge, fasting blood glucose levels were measured andthen mice were injected via tail-vein with a 30 g/L solution of glucoseat a dose of 200 mg/kg. Blood glucose was then monitored every 15minutes for 2 hours.

O. Pancreas Removal and Insulin Quantification

After 174 days, mice treated with human cells encapsulated inTMTD-alginate were euthanized and the pancreas of each mouse removed.Each pancreas was weighed and then placed into vial with a stainlesssteel ball while keeping samples frozen in liquid nitrogen. A volume of3 ml of acid ethanol was added to each vial and samples were homogenizedon a GenoGrinder at 1000 rpm at 1 min increments until tissue waspulverized. Sample vials are held by aluminum blocks that can be placedin liquid nitrogen between each cycle to keep it cold. Vials were thencentrifuged at 2400 rpm at 4° C. for 30 min. The supernatant (nowcontaining insulin) was removed and stored, while the vial is filledwith more acid ethanol and vortexed. The vials were left overnightshaking at 4° C. Again, vials were centrifuged at 2400 rpm at 4° C. for30 min and the supernatant was collected and added to the previouslystored supernatant. Acid ethanol was again added to the vials, vortexed,incubated overnight, centrifuged, and supernatant collected andcombined. Supernatant solution was evaporated using a Genevac EZ-2 plus.Samples were stored at −80° C. until used. Prior to insulinquantification, samples were resuspended in PBS and quantified using amouse insulin ELISA kit (Alpco catalog #: 80-INSMS-E10) according tomanufacturer's instructions. This same procedure was repeated forhealthy, wild type C57BL/6 mice and a STZ treated C57BL/6 mice.

P. Statistical Analysis

Data are expressed as mean±SEM, and N=5 mice per time point and pertreatment group. For Rat studies N=3 per treatment. These sample sizeswere chosen based on previous literature. All animals were included inanalyses except in instances of unforeseen sickness or morbidity. Animalcohorts were randomly selected. Investigators were not blind toperformed experiments. FACS data was analyzed for statisticalsignificance either by unpaired, two-tailed t-test, or one-way ANOVAwith Bonferroni multiple comparison correction, unless indicatedotherwise, as implemented in GraphPad Prism 5; *: p<0.05, **: p<0.001,and ***: p<0.0001.

Quantified data shown are group mean values±SEM.

II. Results

A. TMTD Alginate Mitigates Immunological Responses to Encapsulated HumanCells.

It has been recently demonstrated that microsphere size can have abeneficial impact on resisting immunological responses to implantedalginates, with spheres of 1.5 mm diameters and larger and TMTDalginates, mitigating fibrotic responses in both rodents and non-humanprimates (Veiseh et al. in press and Vegas et al. submitted). Thechemical structure of TMTD is shown in FIG. 13.

To evaluate the immune responses to these spheres, encapsulated humancells were implanted IP into C57BL/6 mice and were retrieved after 14days. Cells associated with the outside of the spheres were isolated andanalyzed by FACS (FIGS. 14 and 15). Statistically significant lowerlevels of macrophages, neutrophils, B cells, and CD8+ T cells weremeasured for TMTD alginate encapsulated human cells (formulation 3)compared to SLG20 controls (formulation 1, 2). Implants retrieved after80-90 days in STZ-C57BL/6J mice revealed that TMTD alginate spheres hadmuch lower levels of fibrotic deposition. Immunofluorescence staining ofthese retrieved spheres for cellular deposition (DAPI, F-actin) andmyofibroblasts (α-SMA) showed significantly lower levels of cellulardeposition on TMTD alginate spheres. Proteomic analysis of these proteinextracts detected 18 collagen isoforms, and 10 out of the 18 detectedcollagen proteins were significantly reduced in TMTD alginatetransplants further showing that these materials are able to mitigatefibrotic responses (FIG. 16).

Western blot quantification of α-SMA protein extracted from theretrieved implants is consistent with lower fibrosis levels on TMTDspheres (FIG. 10).

Finally, consistent with these results, histological processing andimmunostaining of TMTD encapsulated human clusters retrieved after over90 days from STZ-C57BL/6 mice revealed cell clusters with positiveco-localized staining of mature human cell markers human insulin andc-peptide. Minimal to no co-localized staining was observed betweenhuman c-peptide and glucagon or human insulin and glucagon, consistentwith the human cells retaining their differentiation state through theentire study.

The ability of TMTD alginate spheres to provide immunoisolation of theencapsulated human cells was next characterized. Freeze-fracturecryogenic scanning electron microscopy (cryo-SEM) of the spheres displaya heterogeneous pore structure with pore sizes ranging from sub-micronto 1-3 μm in size, a range capable of preventing permeation by cells andlarge proteins. Intravital imaging of transplanted spheres after 14 daysin B6.129S6-Ccr6tm1(EGFP)Irw/J mice (where T, B, and dendritic cellsexpress EGFP) showed localization of CCR6+ cells to regions of spherescontaining human cells, but an inability of these cells to make contactand initiate cytotoxic events.

B. Encapsulation of Human Cells with Triazole-Thiomorpholine Dioxide(TMTD) Alginate Enables Glycemic Correction in STZ-C57BL/6J.

To investigate the potential of microencapsulation of human cells toprovide glycemic correction, cells were used in three differentformulations: (1) 500 μm alginate microcapsules conventionally used forislet encapsulation (Lim et al., Science 210:908-910 (1980); Calafioreet al., Diabetes Care 29:137-138 (2006)), (2) 1.5 mm alginate spheres(Veiseh et al. in press), and (3) 1.5 mm TMTD alginate spheres. Each ofthese formulations was transplanted in streptozotocin (STZ) treatedC57BL/6J mice at three different doses of human cell clusters andevaluated for their ability to restore normoglycemia. Naked,non-encapsulated human cells are unable to provide glycemic correctionin this diabetic model regardless of implantation site.

Encapsulation into 500 j m barium alginate microcapsules is a commonlyimplemented formulation for islet transplantation (Lim et al., Science210, 908-910 (1980); Calafiore, et al., Diabetes Care 29:137-138,(2006)). Mice transplanted with human cells encapsulated in 500 μmmicrocapsules showed the lowest levels of glycemic control, with onlythe highest dose of transplanted clusters able to restore normoglycemiafor 15 days. Human cells encapsulated in 1.5 mm alginate spheresperformed better than the 500 μm microcapsule formulation withnormoglycemia maintained for 20-30 days for the two higher doses,consistent with earlier results obtained using rat islets (Veiseh et al.in press). Sustained normoglycemia was achieved for over 70 days with1.5 mm TMTD alginate spheres at all three doses tested. Robust humanc-peptide levels were measured at 21, 43, and 63 days during the courseof this study, consistent with human cell function, with TMTD alginatespheres showing the highest levels of human c-peptide.

C. Encapsulated Human Cells Support Sustained Normoglycemia and GlucoseResponsiveness in STZ-C57BL/6J.

To evaluate the capacity of TMTD alginate encapsulated human celltransplants to sustain normoglycemia, a cohort of transplanted diabeticmice was tracked for 6 months. Transplanted mice successfully maintainedglycemic correction over the 6-month period, and 5 closely matched theblood glucose levels of wild type C57BL/6J mice tracked over a similarperiod. In addition, robust human c-peptide levels over 100 pmol/L wererecorded at multiple points throughout the study. A glucose challengewas also performed on these mice 150 days post-transplantation, andencapsulated human cells restored normoglycemia comparably to wild typemice. Host pancreas insulin levels for each cohort were also analyzed toconfirm the successful STZ treatment and a lack of endogenous pancreascell regeneration. Human cells implants retrieved after 6 monthsdisplayed no signs of fibrotic overgrowth, with little collagenous andcellular deposition evident on the capsule. Since spheres retrievedafter 3 months showed minimal levels of fibrosis, this indicates thatTMTD alginate mitigates immunological responses by altering the immunerecognition/activation kinetics.

The results show that encapsulated human cells can achieveglucose-responsive, long-term glycemic correction (over 170 days) in animmune-competent diabetic animal with no immunosuppression. This resultwas accomplished by implementing a novel TMTD alginate formulation thatmitigates immunological responses to human cell implants, effectivelydelaying the fibrotic deposition that leads to implant tissue necrosis.This formulation provided sufficient immunoprotection to enablelong-term glycemic correction, in spite of the xenogeneic stimulationthat these human cells manifest in an immunocompetent rodent recipient.These result support the expectation that human cells encapsulated inthe disclosed modified alginates can provide insulin independence forpatients suffering from type 1 diabetes. These result support theexpectation that human cells encapsulated in the disclosed modifiedalginates can provide products produced by the encapsulated cells topatients for long periods of time.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. An implant that is not a living tissue, wherein all or aportion of the implant is coated with a modified alginate comprising oneor more covalently modified monomers defined by Formula I

wherein X is oxygen, sulfur, or NR₄, R₁ is, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, aroxy, substituted aroxy,alkylthio, substituted alkylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, sulfonyl, substitutedsulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptide group, wherein R₁is, independently in at least one covalently modified monomer

or —R₆—R^(b) wherein for compounds of Formula X and XII z is an integerfrom 0 to 5, k is an integer from 1 to 10, X_(d) is absent, O or S, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, and R₁₇ are independently hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,alkoxy, substituted alkoxy, aroxy, substituted aroxy, alkylthio,substituted alkylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, sulfonyl, substituted sulfonyl,sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptide group, whereinR^(a) and R^(c) are independently alkoxy, alkylamino, dialkylamino,hydroxy, alkenyl, alkynyl, substituted alkyl, substituted alkenyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, substituted alkoxy, aroxy, substituted aroxy, alkylthio,substituted alkylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, amino acid, poly(ethylene glycol), peptide, or polypeptidegroup, or together with the carbon atom to which they are attached, forma 3- to 8-membered unsubstituted or substituted carbocyclic,heterocyclic ring or

wherein R₈, R₉, or both are, independently, hydrogen, alkyl, substitutedalkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxy, alkenyl,alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl,carbonyl, substituted carbonyl, carbinol,

wherein R₃₁, R₃₂, R₃₃, R₃₄, R₃₅, and R₃₆ are, independently, hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, aroxy, substituted aroxy,alkylthio, substituted alkylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, amino acid, poly(ethylene glycol), peptide, orpolypeptide group, wherein y is an integer from 0 to 11, wherein R^(e)is independently alkoxy, alkylamino, dialkylamino, hydroxy, alkenyl,alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, substitutedalkoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,arylthio, substituted arylthio, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, and polypeptide group, or togetherwith the carbon atom to which they are attached, form a 3- to 8-memberedunsubstituted or substituted carbocyclic or heterocyclic ring, whereinR₁₈, R₁₉, R₂₀, R₂₁, R₂₂, and R₂₃ are independently C, O, N, or S,wherein the bonds between adjacent R₁₈ to R₂₃ are double or singleaccording to valency, and wherein R₁₈ to R₂₃ are bound to none, one, ortwo hydrogens according to valency, and wherein R₂₄ is independently—(CR₂₅R₂₅)_(p)— or —(CR₂₅R₂₅)_(p)—X_(b)—(CR₂₅R₂₅)_(q)—, wherein p and qare independently integers from 0 to 5, wherein X_(b) is absent, —O—,—S—, —SO₂—, or NR₄′, wherein each R₂₅ is independently absent, hydrogen,═O, ═S, —OH, —SH, —NR₄′, wherein R₄′ is alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, R₈and R₉ are not both hydrogen, wherein at least one R^(a) or R^(c) isdefined by Formula XIII, wherein Y₁ and Y₂ independently are hydrogen or—PO(OR₅)₂, or Y₂ is absent, and Y₁, together with the two oxygen atomsto which Y₁ and Y₂ are attached form a cyclic structure as shown inFormula II

wherein R₂ and R₃ are, independently, hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptide group, or R₂ andR₃, together with the carbon atom to which they are attached, form a 3-to 8-membered unsubstituted or substituted carbocyclic or heterocyclicring, and R₄ and R₅ are, independently, hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptide group, and for—R₆—R^(b), R₆ is alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, sulfonyl, substitutedsulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptide group, and whereinR_(b) is


2. The implant of claim 1, wherein R₁ is, independently in the one ormore covalently modified monomers,

or —R₆—R^(b), wherein a is an integer between1 and 30, inclusive.
 3. Theimplant of claim 2, wherein R₆ is —CH₂-aryl- or —CH₂—CH₂—(O—CH₂—CH₂)₃—,R₄ is hydrogen, R₈ is hydrogen, methyl, or —CH₂—OH, and R₉ is methyl,—COCH₃, —CH₂—N(CH₂—CH₃)₂,


4. The implant of claim 3, wherein R₈ is hydrogen, and R₉ is


5. The implant of claim 1, wherein R₈ is hydrogen, and R₉ is

methyl, —COCH₃, or —CH₂—N(CH₂—CH₃)₂.
 6. The implant of claim 1, whereinX is oxygen or NR₄, and wherein R₄ is hydrogen, methyl, or —CH₂—CH₃. 7.The implant of claim 1, wherein the one or more covalently modifiedmonomers is


8. The implant of claim 1, wherein Y₁ and Y₂ are hydrogen.
 9. Theimplant of claim 1, wherein the one or more covalently modified monomersare selected from the group consisting of

and combinations thereof.
 10. The implant of claim 1, wherein themodified alginate polymer has a structure according to Formula III

wherein X is oxygen, sulfur, or NR₄, R₁, R₇, R₈, and R₉ are,independently, hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, arylthio,substituted arylthio, carbonyl, substituted carbonyl, carboxyl,substituted carboxyl, amino, substituted amino, amido, substitutedamido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl,substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, amino acid, poly(ethyleneglycol), peptide, or polypeptide group, R₆ is alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid,poly(ethylene glycol), peptide, or polypeptide group wherein Y₁ and Y₂independently are hydrogen or —PO(OR₅)₂, or Y₂ is absent, and Y₁,together with the two oxygen atoms to which Y₁ and Y₂ are attached forma cyclic structure as shown in Formula IV


11. The implant of claim 10, wherein R₁ is, independently,

or —R₆—R^(b), wherein a is an integer from 1 to 30, z is an integer from0 to 5, n is an integer from 1 to 12, m is an integer from 3 to 16, andwherein R^(b) is


12. The implant of claim 10, wherein R₆ is —CH₂-aryl- or—CH₂—CH₂—(O—CH₂—CH₂)₃—, R₇ is hydrogen, R₈ is hydrogen, methyl, or—CH₂—OH, and R₉ is methyl, —COCH₃, —CH₂—N(CH₂—CH₃)₂,


13. The implant of claim 12, wherein R₈ is hydrogen, and R₉ is


14. The implant of claim 12, wherein R₈ is hydrogen, and R₉ is

methyl, —COCH₃, or —CH₂—N(CH₂—CH₃)₂.
 15. The implant of claim 10,wherein X is oxygen or NR₄, wherein R₄ is hydrogen, methyl, or —CH₂—CH₃,and R₁ is —CH₂—CH₂—CH₂—CH₂—OH, —CH₂—CH₂—(O—CH₂—CH₂)_(m)—O—CH₃, where mis an integer from 3 to 16, —CH₂—CH₂—O—CH₂—CH₂—OH, —(CH₂—CH₂)₃—NH—CH₃,


16. The implant of claim 10, wherein Y₁ and Y₂ are hydrogen.
 17. Theimplant of claim 10, wherein the implant is an implantable device, acardiac pacemaker, a catheter, a needle injection catheter, a blood clotfilter, a balloon, a stent, a coil device, a surgical repair mesh, atransmyocardial revascularization device, a percutaneous myocardialrevascularization device, a prosthesis, a heart valve, a tube, avascular implant, a fiber, a hollow fiber, a membrane, a textile, ablood container, a titer plate, an adsorber media, a dialyzer, aconnecting piece, a sensor, a valve, an endoscope, a filter, a pumpchamber, or a hemocompatible medical device.
 18. The implant of claim17, wherein the modified alginate polymer is non-covalently associatedwith the surface of the implant.
 19. The implant of claim 17, whereinthe modified alginate is covalently associated with the surface of theimplant.